\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[greek,ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{Inactivation of SERCA2 Cys674 accelerates aortic aneurysms by
suppressing PPAR\selectlanguage{greek}γ\selectlanguage{english}}



\author[1]{Yumei Que}%
\author[1]{Xi Shu}%
\author[1]{Langtao Wang}%
\author[1]{Sai Wang}%
\author[1]{Pingping Hu}%
\author[1]{Xiaoyong Tong}%
\affil[1]{Innovative Drug Research Center}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background and Purpose Inactivation of Cys674 (C674) in the
sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) by causing the
accumulation of intracellular Ca2+ to activate calcineurin-mediated
nuclear factor of activated T-lymphocytes (NFAT)/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways,
resulted in the phenotypic modulation of smooth muscle cells (SMCs) to
accelerate angiotensin II-induced aortic aneurysm. Our goal was to
investigate the mechanism involved. Experimental Approach We used
heterozygous SERCA2 C674S knock-in (SKI) mice, where half of C674 was
substituted by serine, to represent partial irreversible oxidation of
C674. The aortas of SKI mice and their littermate wild-type mice were
collected for RNA sequencing, cell culture, protein expression,
luciferase activity and aortic aneurysm analysis. KEY RESULTS
Inactivation of C674 inhibited the promoter activity and protein
expression of PPAR\selectlanguage{greek}γ, \selectlanguage{english}which could be reversed by inhibitors of
calcineurin or NF-\selectlanguage{greek}κ\selectlanguage{english}B. Overexpression of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 inhibited the phenotypic
modulation of SKI SMCs. Pioglitazone, the activator of PPAR\selectlanguage{greek}γ, \selectlanguage{english}blocked
the activation of NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B, inhibited SMC phenotypic modulation, and
ameliorated angiotensin II-induced aortic aneurysms in SKI mice.
CONCLUSIONS AND IMPLICATIONS The inactivation of SERCA2 C674 promotes
the development of aortic aneurysm by disrupting the balance between
PPAR\selectlanguage{greek}γ \selectlanguage{english}and NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B. Our study highlights the importance of C674 redox
status in regulating PPAR\selectlanguage{greek}γ \selectlanguage{english}to maintain aortic homeostasis.%
\end{abstract}%



\sloppy


\textbf{Title:}Inactivation of SERCA2 Cys\textsuperscript{674}
accelerates aortic aneurysms by suppressing PPAR\selectlanguage{greek}γ

\selectlanguage{english}\textbf{Running title:} Inactivation of SERCA2
Cys\textsuperscript{674}suppresses PPAR\selectlanguage{greek}γ

\selectlanguage{english}\textbf{Authors}

Yumei Que\textsuperscript{1}, Xi Shu\textsuperscript{1}, Langtao
Wang\textsuperscript{1}, Sai Wang\textsuperscript{1}, Pingping
Hu\textsuperscript{1}, Xiaoyong Tong\textsuperscript{1*}

\textbf{Affiliations}

1. School of Pharmaceutical Sciences, Chongqing University, Chongqing,
401331, China

To whom correspondence should be addressed: Xiaoyong Tong,
xiaoyongtong@cqu.edu.cn

\textbf{Word count} (excluding Methods, Abstract, References, Figure
legends): 3937

\textbf{Illustrations:} 8 figures, 1 table.

\textbf{Abstract}

\textbf{Background and Purpose}

Inactivation of Cys\textsuperscript{674} (C674) in the
sarcoplasmic/endoplasmic reticulum Ca\textsuperscript{2+} ATPase 2
(SERCA2) by causing the accumulation of intracellular
Ca\textsuperscript{2+} to activate calcineurin-mediated nuclear factor
of activated T-lymphocytes (NFAT)/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways, resulted in the
phenotypic modulation of smooth muscle cells (SMCs) to accelerate
angiotensin II-induced aortic aneurysm. Our goal was to investigate the
mechanism involved.

\textbf{Experimental Approach}

We used heterozygous SERCA2 C674S knock-in (SKI) mice, where half of
C674 was substituted by serine, to represent partial irreversible
oxidation of C674. The aortas of SKI mice and their littermate wild-type
mice were collected for RNA sequencing, cell culture, protein
expression, luciferase activity and aortic aneurysm analysis.

\textbf{KEY RESULTS}

Inactivation of C674 inhibited the promoter activity and protein
expression of PPAR\selectlanguage{greek}γ, \selectlanguage{english}which could be reversed by inhibitors of
calcineurin or NF-\selectlanguage{greek}κ\selectlanguage{english}B. Overexpression of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 inhibited the phenotypic
modulation of SKI SMCs. Pioglitazone, the activator of PPAR\selectlanguage{greek}γ, \selectlanguage{english}blocked
the activation of NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B, inhibited SMC phenotypic modulation, and
ameliorated angiotensin II-induced aortic aneurysms in SKI mice.

\textbf{CONCLUSIONS AND IMPLICATIONS}

The inactivation of SERCA2 C674 promotes the development of aortic
aneurysm by disrupting the balance between PPAR\selectlanguage{greek}γ \selectlanguage{english}and NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B. Our
study highlights the importance of C674 redox status in regulating PPAR\selectlanguage{greek}γ
\selectlanguage{english}to maintain aortic homeostasis.

\textbf{KEY WORDS}

SERCA2, aortic aneurysm, smooth muscle, phenotypic modulation, PPAR\selectlanguage{greek}γ,
\selectlanguage{english}NFAT, NF-\selectlanguage{greek}κ\selectlanguage{english}B.

\textbf{Summary}

\textbf{`What is already known'}

The inactivation of SERCA2 Cys\textsuperscript{674} by causing the
accumulation of intracellular Ca\textsuperscript{2+} to activate
calcineurin-mediated NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways, results in SMC phenotypic
modulation to accelerate aortic aneurysm.

\textbf{`What this study adds'}

\begin{itemize}
\tightlist
\item
  Inactivation of SERCA2 Cys\textsuperscript{674} causes the imbalance
  between PPAR\selectlanguage{greek}γ \selectlanguage{english}and NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B in SMCs, leads to the sustained
  activation of NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B.
\item
  Activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}could restore this balance, thus suppresses SMC
  phenotypic modulation and aortic aneurysm formation caused by the
  inactivation of SERCA2 Cys\textsuperscript{674}.
\end{itemize}

\textbf{`Clinical significance'}

\begin{itemize}
\tightlist
\item
  Increase levels of ROS irreversibly oxidize Cys\textsuperscript{674}in
  SERCA2 to promote the development of aortic aneurysm by suppressing
  PPAR\selectlanguage{greek}γ.
\selectlanguage{english}\item
  Activating PPAR\selectlanguage{greek}γ \selectlanguage{english}contributes to controlling aortic aneurysm by
  restricting SMC phenotypic modulation.
\item
  PPAR\selectlanguage{greek}γ \selectlanguage{english}and SERCA2 may be potential therapeutic targets for aortic
  aneurysm.
\end{itemize}

\textbf{Abbreviations}

C674, Cys\textsuperscript{674}; Col I, collagen type I; Col III,
collagen type III; CsA, cyclosporin A; DAPI,
4',6-diamidino-2-phenylindole; FPKM, expected number of fragments per
kilobase of transcript sequence per millions base pairs sequenced; HE,
hematoxylin/eosin; ICAM1, intercellular adhesion molecule 1;
LDLR\textsuperscript{-/-}, LDL receptor-deficient; MYOCD, myocardin;
NFAT, nuclear factor of activated T-lymphocytes; OPN, osteopontin; PDTC,
pyrrolidinedithiocarbamic acid; S674, Ser\textsuperscript{674}; SERCA,
sarco/endoplasmic reticulum calcium ATPase; SKI, SERCA2 C674S knock-in;
SMCs, smooth muscle cells; VCAM1, vascular cell adhesion molecule 1;
VVG, Verhoeff-Van Gieson; WT, wild-type

\textbf{1 \textbar{} Introduction}

Aortic aneurysm is a fatal vascular disease, lack of effective drug
treatment, surgery is the only choice. Most aortic aneurysms are
asymptomatic before catastrophic complications such as dissection or
rupture occur. Although surgery can stabilize large aneurysms, the
recurrence rate and mortality rate are still high. Therefore, it is
urgent to clarify the molecular mechanism of the occurrence and
development of aortic aneurysm and determine the target of intervention.

Smooth muscle cells (SMCs) located in the media of aorta are key to the
vascular remodeling of aortic aneurysm. Most SMCs are in the state of
differentiation under physiological conditions to maintain the
elasticity and tensile strength of the vascular wall. However, SMCs have
significant phenotypic plasticity and can quickly adapt to environmental
fluctuation signals. The transition of SMCs from a differentiated state
to a dedifferentiated state is called phenotypic modulation,
characterized by enhanced proliferation, migration, and expression of
fibrotic and inflammatory proteins, which is the key cellular basis of
aortic aneurysm (Ailawadi et al., 2009). Myocardin (MYOCD) is a
differentiation marker of SMCs, and its deletion in SMCs causes severe
aortic aneurysm (Tang et al., 2008). Matrix cytokine \emph{osteopontin
(OPN)} is a marker of dedifferentiation of SMCs, and its serum level
predicts the progression of aortic aneurysm (Golledge et al., 2007).
Collagen type I and type III (\emph{Col I} , \emph{Col III} ) correlate
well with the size of aortic aneurysm, and they are also SMC
dedifferentiation markers (Meng et al., 2014; Wang et al., 2012a)
.\emph{MMP2} degrades elastin and collagen (mainly Col IV in basal
laminae), destroys vascular integrity, critical in angiotensin
II-induced aortic aneurysm (Wang et al., 2012b). NF-\selectlanguage{greek}κ\selectlanguage{english}B is a
transcriptional factor and its activation favors its nuclear
translocation to up-regulate the gene expression of MMP2, \emph{vascular
cell adhesion molecule 1 (VCAM1)} and \emph{intercellular adhesion
molecule 1 (ICAM1)} , promotes SMC proliferation, migration, and
inflammation (de Winther et al., 2005; Monaco et al., 2004; Tsai et al.,
2017). NF-\selectlanguage{greek}κ\selectlanguage{english}B is up-regulated in the aortic aneurysm wall, and inhibition
of NF-\selectlanguage{greek}κ\selectlanguage{english}B decreases the size of aortic aneurysm (Miyake et al., 2007).

\emph{Sarco/endoplasmic reticulum calcium ATPase (SERCA)} is a key
enzyme to maintain calcium homeostasis by taking up
Ca\textsuperscript{2+} from cytosol to sarcoplasmic reticulum and
endoplasmic reticulum. SERCA2 is the main subtype of SERCA in the
vasculature, and the \emph{S} -glutathiolation of the amino acid residue
Cys\textsuperscript{674} (C674) is key to increase the activity of
SERCA2 under physiological conditions (Adachi et al., 2004; Tong et al.,
2008), but this post-translational protein modification is prevented by
the irreversible oxidation of C674 thiol in pathological situations
characterised by high levels of ROS, such as aging, diabetes mellitus,
atherosclerosis, and hypertension (Adachi et al., 2004; Liu et al.,
2020; Qin et al., 2013; Ying et al., 2008), which are risk factors for
aortic aneurysm. We have reported that the irreversible oxidation of
C674 occurred broadly in mouse and human aortic aneurysms (Que et al.,
2020). We used heterozygous SERCA2 C674S knock-in (SKI) mice, where half
of C674 was substituted by serine, to represent partial irreversible
oxidative inactivation of C674 under pathological conditions. The
inactivation of C674 by causing the accumulation of intracellular
Ca\textsuperscript{2+} to activate Ca\textsuperscript{2+}-dependent
calcineurin that promoted the nuclear translocation of nuclear factor of
activated T-lymphocytes (NFAT) and NF-\selectlanguage{greek}κ\selectlanguage{english}B, therefore down-regulated the
expression of MYOCD, while up-regulated the expression of OPN, MMP2, Col
I, Col III, and VCAM1, accelerated SMC proliferation, migration and
macrophage adhesion to SMCs, and exacerbated angiotensin II-induced
aortic aneurysm (Que et al., 2020). We hypothesize that inactivation of
SERCA2 C674 not only activates NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B, but also inhibits the
protective factors that restrict the phenotypic modulation of SMCs, thus
accelerating the occurrence of aortic aneurysm. This study was designed
to elucidate the downstream targets of C674 in regulating SMC phenotypic
modulation and aortic aneurysm.

\textbf{2 \textbar{} Methods}

\textbf{2.1 \textbar{} Animals}

All animal care and study protocols complied with the guidelines of the
ethical use of animals and were approved by the Animal Care and Use
Committee (Third Military Medical University, Chongqing, China). All
efforts were made to minimize animal suffering and reduce the number of
animals used. Animal studies are reported in compliance with the ARRIVE
guidelines and with the recommendations made by the \emph{British
Journal of Pharmacology (BJP)} (Kilkenny et al., 2010; McGrath \&
Lilley, 2015). Experimental protocols and design adhere to
\emph{BJP}guidelines (Curtis et al., 2015).

Mice were bred and housed in the laboratory animal room at Chongqing
University under specific pathogen-free conditions. Mice were kept in
open polypropylene cages with clean chip bedding. The animal room was
maintained at a controlled temperature (22 \selectlanguage{ngerman}± 3 °C) and a 12 hr cycle of
light and dark. Five mice in each cage were free to drink water and were
fed on a regular diet (0.3--0.8\% sodium, 1.0--1.8\% calcium, 0.6--1.2\%
phosphorus, Beijing Keao Xieli Feed Corporation, China) unless otherwise
indicated. To obtain tissues, all mice were killed by i.p. injection
with 2,2,2-tribromoethanol (250 mg·kg\selectlanguage{english}\textsuperscript{-1},
Sigma-Aldrich, Cat\# T48402).

\textbf{2.2 \textbar{} SERCA2 C674S knock-in mouse construct}

All mice used in this study were of C57BL/6J background (\emph{RRID:
IMSR\_JAX:000664} ) and were originally obtained from The Jackson
Laboratory (Bar Harbor, Maine, USA). The construct of the SKI mouse
(\emph{RRID: MGI:5780876} ) was generated as previously described
(Thompson et al., 2014). Briefly, the genetic variation is TGT at C674
to TCC (Ser\textsuperscript{674} {[}S674{]}) in exon 14 of SERCA2. The
presence of the C674S mutation was verified by sequencing of both
genomic DNA and cDNA from heart. Homozygous SKI mice died before birth,
so only heterozygous SKI mice that express 50\% of C674 and 50\% of S674
were used in this study, and their littermate wild-type (WT) mice
without S674 were used as controls. For aneurysm studies, SKI mice were
mated with LDL receptor-deficient (LDLR\selectlanguage{english}\textsuperscript{-/-}, The
Jackson Laboratory, \emph{RRID: IMSR\_JAX:002207)} mice susceptible to
aortic aneurysms to obtain SKI/LDLR\selectlanguage{english}\textsuperscript{+/-} mice, which
were further mated with LDLR\selectlanguage{english}\textsuperscript{-/-} mice to obtain
SKI/LDLR\selectlanguage{english}\textsuperscript{-/-} mice. All mice used for aneurysm studies
were derived from mating between SKI/LDLR\selectlanguage{english}\textsuperscript{-/-} mice and
LDLR\selectlanguage{english}\textsuperscript{-/-} mice, and the littermate
LDLR\selectlanguage{english}\textsuperscript{-/-} mice acted as controls.

\textbf{2.3 \textbar{} RNA sequencing and bioinformatics}

Aorta of 4-month-old male from LDLR\selectlanguage{english}\textsuperscript{-/-} or
SKI/LDLR\selectlanguage{english}\textsuperscript{-/-} mice were homogenized in Trizol for RNA
isolation using the Direct-zol\selectlanguage{english} RNA MiniPrep Kit (Zymo Research, Irvine,
CA). RNA sequencing was done in Novogene Bioinformatic Technology Co.
Ltd (Beijing, China). Briefly, construction of RNA sequencing library
was performed on an Illumina(r) Hiseq platform. Paired-end clean reads
were aligned to the mouse genome (GRCm38, RRID:SCR\_002344), using
TopHat v2.0.12. The reads numbers mapped to each gene were quantified
using HTSeq v0.6.1. The expected number of fragments per kilobase of
transcript sequence per millions base pairs sequenced (FPKM) of each
gene was calculated based on the length of the gene and reads count
mapped to this gene that reflects the relative gene expression level.
Differentially expressed genes between LDLR\selectlanguage{english}\textsuperscript{-/-} and
SKI/LDLR\selectlanguage{english}\textsuperscript{-/-} mice were analysed using DESeq R package
(1.18.0). Gene set enrichment analysis was performed on differentially
expressed genes (altered \textgreater{} 1.5 fold) to test for enrichment
of specific ontologies (Subramanian et al., 2005), and the Kyoto
encyclopedia of genes and genomes (http://www.genome.jp/kegg/) was used
to perform pathway analysis.

\textbf{2.4 \textbar{} Real-time quantitative PCR}

Total RNA was isolated from aorta using TRIzol reagent (Invitrogen,
Cat\# 15596026) and retro-transcribed to cDNA using cDNA PCR kit (Takara
Bio Inc., Cat\# RR037Q). Real-time quantitative PCR was performed using
SYBR-Green-based detection (Takara Bio Inc., Cat\# RR420L) with primers
commercially synthesized (Sangon Biotech, China). The primer sequences
from 5' to 3' are: 1) PPAR\selectlanguage{greek}γ\selectlanguage{english}2 forward, TCGCTGATGCACTGCCTATG, PPAR\selectlanguage{greek}γ\selectlanguage{english}2
reverse, GAGAGGTCCACAGAGCTGATT; 2) internal control \selectlanguage{greek}β-\selectlanguage{english}actin forward,
AGAGGGAAATCGTGCGTGAC, \selectlanguage{greek}β-\selectlanguage{english}actin reverse, CAATAGTGATGACCTGGCCGT. Real time
quantitative PCR was performed using the following cycling conditions:
denaturation, annealing, and extension at 95\selectlanguage{ngerman}°C, 57°C and 72°C for 10 s,
30 s, and 10 s, respectively, for 40 cycles. Relative expression of
PPAR\selectlanguage{greek}γ\selectlanguage{english}2 was analysed using the comparative Ct method
(2\textsuperscript{-ΔΔCt}\selectlanguage{english}).

\textbf{2.5 \textbar{} Aortic SMC culture}

Aortic SMCs were isolated from 8-week-old male WT and SKI mice as
previously described (Tong et al., 2015) and cultured in DMEM
supplemented with 10\% FBS (ExCell Bio, Cat\# FSP500), 100 U/mL
penicillin and 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL streptomycin at 37\selectlanguage{ngerman}°C in a humidified atmosphere
containing 5\% CO\textsubscript{2}. SMC phenotype was confirmed by
\selectlanguage{greek}α-\selectlanguage{english}smooth muscle actin immunostaining. Cells from passages 3 to 8 were
used. All SMCs used were isolated from C57BL/6J background due to the
SMCs from LDLR\textsuperscript{-/-} background were infeasible for
further studies after subculture. Using different genetic background for
mechanism study is commonly used in literature (Nakao et al., 2017). In
some experiments, SMCs were administrated with pioglitazone (10 \selectlanguage{greek}μ\selectlanguage{english}M, MCE,
Cat\# U72107), or pyrrolidinedithiocarbamic acid (PDTC, ammonium salt,
10 \selectlanguage{greek}μ\selectlanguage{english}M, Macklin, Cat\# A800469) for 48 hr, before being collected for
Western blot analysis or cell function studies. DMSO served as a solvent
control, and its final concentration was less than 0.1\%.

\textbf{2.6 \textbar{} Western blot}

Aortas or SMCs cultured in 0.2\% FBS DMEM overnight were lysed in RIPA
buffer (Enogene, Cat\# E1WP106). Experimental details of Western blots
are in accordance with \emph{BJP} guidelines (Alexander et al., 2018).
In some experiments, protein was extracted from the nucleus and
cytoplasm by using the protein extraction kit (Beyotime Biotechnology,
Cat\# P0027). Proteins were separated by SDS-PAGE electrophoresis using
standard methods, transferred to PVDF membrane, and immunoblotted with
specific antibodies overnight at 4 \selectlanguage{ngerman}°C against PPAR\selectlanguage{greek}γ \selectlanguage{english}(detect both PPAR\selectlanguage{greek}γ\selectlanguage{english}1
and PPAR\selectlanguage{greek}γ\selectlanguage{english}2, Engene, Cat\# E2A6073, 58 kDa, \emph{RRID:AB\_2861246} ),
MYOCD (Abcam, Cat\# Ab107301,100 kDa, \emph{RRID:AB\_11128102} ), OPN
(Proteintech, Cat\# 22952-1-AP, 44 kDa, \emph{RRID:AB\_2783651} ), MMP2
(Proteintech, Cat\# 10373-2-AP, 64 kDa, \emph{RRID:AB\_2250823} ), Col I
(Engene, Cat\# E110154C, 139 kDa, \emph{RRID:AB\_2861247} ), Col III
(Abcam, Cat\# Ab7778, 139 kDa, \emph{RRID:AB\_306066} ), p65NF-\selectlanguage{greek}κ\selectlanguage{english}B
(Engene, Cat\# E011014, 65 kDa, \emph{RRID:AB\_2861239} ),
phosphorylated p65NF-\selectlanguage{greek}κ\selectlanguage{english}B (Engene, Cat\# E1A2006V, 65 kDa,
\emph{RRID:AB\_2861248} ), VCAM1 (Engene, Cat\# E90279, 72 kDa,
\emph{RRID:AB\_2861237} ), ICAM1 (Engene, Cat\# E301187, 82 kDa,
\emph{RRID:AB\_2861238} ), NFAT4 (Abcam, Cat\# Ab93628, 117 kDa,
\emph{RRID:AB\_10714571} ), GAPDH (Engene, Cat\# E1C604, 37 kDa,
\emph{RRID: AB\_2814765} ), Histone H3 (Engene, Cat\# E11-0434B, 17 kDa,
\emph{AB\_2861240} ), followed by incubation with HRP-conjugated goat
anti-rabbit secondary antibody (Sino Biological Inc., Cat\# SSA003,
\emph{RRID:AB\_2814815} ) 1 hr at room temperature. Proteins were
visualized with an X-ray film system (Fujifilm, Japan) or ChemiDoc
Touch System (Bio-Rad, USA). Band density was quantified by NIH ImageJ
software (\emph{RRID:SCR\_003070} , https://imagej.net/) and normalized
to GAPDH or Histone H3.

\selectlanguage{greek}\textbf{2.7 \textbar{} Construction and detection of luciferase reporter
of mouse PPARγ2 promoter}\selectlanguage{english}

There are two NFAT binding (TTTTCC) sites in the human PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter
(distal -299 to -294 bp and proximal -146 to -141 bp) to fully regulate
its expression in adipocytes (Kim et al., 2010) and hepatoma cells (Yang
et al., 2003). However, these two NFAT binding sites haven't been
reported in mouse PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter. We compared the sequence of mouse and
human PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoters from -2000 to +45 bp and found that there were
two potential NFAT binding sites in mouse PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter, of which -395
to -390 bp (TTTTCT) was homologous to human distal NFAT site, and -238
to -233 bp (TTTTCC) was homologous to human proximal NFAT site. These
two NFAT binding sites also covered the NF-\selectlanguage{greek}κ\selectlanguage{english}B binding sequence (TTTCC).
Mouse liver genomic DNA was extracted as template to amplify the
promoter sequence of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 from -630 to +45 bp that contains the
putative NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B binding sites and its 5'- untranslated region
(GenBank, AY243584.1) with forward primer
(5'AGAACATTTCTCTATCGATA\emph{GGTACC} TAGAATTTGGATAGCAGTAAC3') containing
Kpn I digestion site and reverse primer containing Hind III digestion
site (5'ACCAACAGTACCGGAATGCC\emph{AAGCTT} AACAGCATAAAACAGAGATTTG3').

The amplified DNA fragments were digested by Kpn I (BioLabs, Cat\#
R0142S) and Hind III (BioLabs, Cat\# R0104S), and purified. The pLG4.19
basic plasmid vector (Promega, Cat\# E6741) containing firefly
luciferase sequence was digested with Kpn I and Hind III to obtain the
linearized vector. A PPAR\selectlanguage{greek}γ\selectlanguage{english}2-pLG4.19 plasmid containing mouse PPAR\selectlanguage{greek}γ\selectlanguage{english}2
promoter (-630 to +45 bp) and firefly luciferase was obtained by mixing
purified PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter fragment, linearized pLG4.19 and assembly
reagent (TransGene Biotech, Cat\# CU101) for 15 min at 50 \selectlanguage{ngerman}°C. PCR and
sequencing confirmed that the PPAR\selectlanguage{greek}γ\selectlanguage{english}2-pLG4.19 firefly luciferase plasmid
was constructed correctly. Large scale amplification of PPAR\selectlanguage{greek}γ\selectlanguage{english}2-pLG4.19
plasmid was carried out in TransT1 Escherichia coli. For mouse PPAR\selectlanguage{greek}γ\selectlanguage{english}2
promoter activity assay, 1 \selectlanguage{greek}μ\selectlanguage{english}g PPAR\selectlanguage{greek}γ\selectlanguage{english}2-pLG4.19 firefly luciferase and 100
ng pRL-TK renilla luciferase plasmid control (Promega, Cat\# E2241) were
co-transfected into SMCs (8 \selectlanguage{ngerman}× 10\textsuperscript{5} cells per 60 mm
dish) using SuperFect Transfection Reagent (Qiagen, Germany, Cat\#
301305) as per manufacturer's recommendations. Luciferase activities
were measured using the Dual-Luciferase reporter assay kit (Beyotime
Biotechnology, Cat\# RG027) 72 hr after transfection. The ratio of
firefly to renilla luciferase luminescence was calculated as indicative
of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter activity. In some experiments, SMCs were treated
cyclosporin A (CsA, 1 \selectlanguage{greek}μ\selectlanguage{english}M) or PDTC (10 \selectlanguage{greek}μ\selectlanguage{english}M) for 24 hr before measuring the
activity of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter.

\selectlanguage{greek}\textbf{2.8 \textbar{} Overexpression of PPARγ2 in SMCs}\selectlanguage{english}

The PPAR\selectlanguage{greek}γ\selectlanguage{english}2 plasmids (ORIGENE, Cat\# MR208132) or its control CMV-GFP
plasmids (ORIGENE, Cat\# PS100040) were transfected into SKI SMCs
according to Effectene\selectlanguage{ngerman}® Transfection Reagent Handbook (Qiagen,
Cat\#301305). The fluorescence intensity of GFP was observed by
fluorescence microscope (Leica, DMi8) to evaluate the transfection
efficiency. SMCs were collected 48 hr later for protein analysis and
cell function studies.

\textbf{2.9 \textbar{} Cell proliferation assay}

A tetrazolium-based non-radioactive proliferation assay kit (Quick Cell
Proliferation Assay Kit II, BioVision, Cat\# K301-500) was used to
determine the cell number according to the manufacturer's protocol (Tong
et al., 2016). SKI SMCs were seeded in 96-well plate at a density of 5 ×
10\textsuperscript{3} cells per well in DMEM supplemented with 0.2\% FBS
overnight. Cell proliferation was stimulated by medium supplemented with
10\% FBS, and 0.2\% FBS medium was used as a control. PDTC (10 \selectlanguage{greek}μ\selectlanguage{english}M) or
pioglitazone (10 \selectlanguage{greek}μ\selectlanguage{english}M) was added to the culture media. DMSO served as a
solvent control. Culture medium with high or low serum was changed
daily. The cell number in each well was determined 72 hr later.

\textbf{2.10 \textbar{} Wounded monolayer migration assay}

SKI SMCs were pretreated with PDTC (10 \selectlanguage{greek}μ\selectlanguage{english}M) or pioglitazone (10 \selectlanguage{greek}μ\selectlanguage{english}M) for
48 hr before migration assay (Tong et al., 2016). DMSO acted as solvent
control. 10\textsuperscript{6} cells/well in 12-well plates were seeded
in 0.2\% FBS DMEM overnight to reach confluence, then scratch wounds
were applied to SMC monolayer with a pipette tip. Immediately after
scratching, the cells were treated with 10\% FBS DMEM to stimulate cell
migration. Photographs were taken at 0 hr and 6 hr at three fixed
locations along the scratch with a light microscope and analyzed using
NIH Image J software.

\textbf{2.11 \textbar{} Macrophage adhesion assay}

Bone marrow-derived mononuclear cells were isolated from the femurs of
WT mice and cultured in high-glucose DMEM supplemented with 10\% FBS and
20\% L929-conditioned medium for 5 days; at this point, adherent
mononuclear cells are macrophages. SKI SMCs were pretreated with PDTC
(10 \selectlanguage{greek}μ\selectlanguage{english}M) or pioglitazone (10 \selectlanguage{greek}μ\selectlanguage{english}M) for 48 hr before macrophage adhesion
assay. DMSO acted as solvent control. For macrophage adhesion assay
(Tong et al., 2016), SMCs were plated in 12-well plate, at a density of
10\textsuperscript{6} cells/well, in 0.2\% FBS DMEM until confluent.
Macrophages (5 \selectlanguage{ngerman}× 10\textsuperscript{5} cells/well) were added to SMCs
and incubated for 1 hr; the media was removed and SMCs washed 3 times
with PBS to remove unbound macrophages. Four images were taken in each
well and the number of bound macrophages was counted per image area.

\textbf{2.12 \textbar{} Angiotensin II-induced aortic aneurysm}

Male WT and SKI mice in LDLR\selectlanguage{english}\textsuperscript{-/-} background at 7 weeks
old (18--22 g) were fed on a Western diet (21\% w/w milk fat, 0.15\% w/w
cholesterol, Beijing Keao Xieli Feed Corporation) for 7 days and then
received subcutaneous implantation of ALZET osmotic pumps (Model 2004,
DURECT Corporation, USA), delivering angiotensin II (1.44
mg*kg\selectlanguage{english}\textsuperscript{-1}*day\selectlanguage{english}\textsuperscript{-1}, dissolved in
saline)(Bhamidipati et al., 2014). Before implantation, mice were given
buprenorphine (0.08 mg*kg\selectlanguage{english}\textsuperscript{-1}, s.c.) as analgesic, and
during pump implantation, they were anaesthetised with isoflurane
(1.5\%) inhalation. The mouse back was depilated with depilatory
ointment. After disinfection with iodophor, the pump was implanted
subcutaneously into the back of the mouse. The incision was sealed with
sterile sutures and disinfected with iodophor. Buprenorphine (0.08
mg*kg\selectlanguage{english}\textsuperscript{-1}, s.c., every 8 hr) was given to the mouse the
first day after surgery. The mice were monitored every day in the first
week after operation. Seven days after angiotensin II infusion, SKI mice
were randomly given pioglitazone
(15\selectlanguage{english} mg*kg\selectlanguage{english}\textsuperscript{-1}*day\selectlanguage{english}\textsuperscript{-1}, suspended in 200
\selectlanguage{greek}μ\selectlanguage{english}L sterile water, \emph{n} = 11) or solvent control sterile water (200
\selectlanguage{greek}μ\selectlanguage{english}L, \emph{n} = 11), and WT mice were given sterile water (200
\selectlanguage{greek}μ\selectlanguage{english}L,\emph{n} = 11). They were given by gavage once a day. Western diet
was applied during the whole experiment. 28 days after the infusion of
angiotensin II, mice were killed and perfused with ice-cold PBS, and
then the connective tissue was removed to expose the whole aorta and its
collateral branches and digital images were acquired for aneurysm
analysis using an Olympus microscope (model SZX16). Aneurysms are
defined as enlargements \textgreater{} 50\% diameter compared to
littermate controls without angiotensin II infusion. The composition of
aneurysms was evaluated according to the type of aneurysm, including the
ruptured aneurysm, unruptured aneurysm, and non-aneurysm. The survival
rate and incidence of aneurysm were monitored and calculated. The number
of aneurysms per mouse was counted. The length of aneurysm was measured
according to the scale with CellSens standard software 1.12
(\emph{RRID:SCR\_014551} , Olympus).

\textbf{2.13 \textbar{} Histology and immunohistochemistry}

Aneurysms were carefully dissected and fixed in 4\% paraformaldehyde
overnight, followed by 30\% sucrose solution for 24 hr before embedding
in optimum cutting temperature compound to prepare serial frozen
sections at a thickness of 7 \selectlanguage{greek}μ\selectlanguage{english}m. Remaining portions of the aorta were
snap-frozen in liquid nitrogen and stored at -80 \selectlanguage{ngerman}°C for protein
analysis. Twenty serial sections of the largest aneurysm for each mouse
were prepared and stained individually with hematoxylin/eosin (HE),
Verhoeff-Van Gieson (VVG) and Masson's trichrome for morphological
analysis, elastin assessment and collagen deposition, respectively. The
area of aneurysm was measured according to the scale with CellSens
standard software. The collagen deposition was scored on a scale of 1 to
4, with 1- \textless{} 25\% area in medium layer was occupied by
collagen, 2--25-50\% collagen deposition, 3--50-75\% collagen
deposition, and 4- \textgreater{} 75\% collagen deposition. Elastin
lamellae were evaluated using a grade of I to IV, with I- intact
internal elastic lamina, II- mild elastin fragmentation, III- severe
elastin digestion, and IV- severe elastin digestion with visible
ruptured sites. The collagen deposition and elastin degradation scores
among different observers blinded to the experimental groups were
comparable; data are expressed as the mean value of different observers'
scores.

\textbf{2.14 \textbar{} Measurement of plasma cholesterol and
triglycerides}

Mice were sacrificed and blood was drawn from the right atrium into
EDTA-containing tubes, for lipid measurements. Plasma was prepared via
centrifugation at 850 ×g for 15 min at 4 °C and stored at -20 °C.
Measurements were carried out using the Infinity Cholesterol and
Triglycerides measurement kits (Thermo Scientific, USA, Cat\# TR13421,
Cat\# TR22421), based on the absorbance of samples normalized to the
absorbance of a known concentration of a calibrator provided with the
kit.

\textbf{2.15 \textbar{} Data and statistical analysis}

The data and statistical analysis comply with the recommendations of
the\emph{BJP} on experimental design and analysis in pharmacology
(Curtis et al., 2018). All studies were designed to generate groups of
equal size, using randomization and blinded analysis. Group size is the
number of independent values, and statistical analysis was carried out
using these independent values. Sample sizes in each group subjected to
statistical analysis were determined based on our previous studies,
preliminary results, and power analysis (Curtis et al., 2015).
Statistical analysis was undertaken only using these independent values
with \emph{n} \selectlanguage{english}[?] 5. Fold change over control was used in Western blot and
gene analysis to avoid the larger variation among different experiments.
The mean values of the control group were normalized to 1. In the
figures, the Y axis shows the ratio of the experimental group to that of
the corresponding matched control values and is labelled as ``fold
matched control values.'' All results were expressed as mean +- SEM.
Statistical analysis was performed using GraphPad Prism 7.00
(\emph{RRID:SCR\_002798} , http://www.graphpad.com/). Unpaired Student's
t test was used to analyse data from two groups. Differences among
multiple groups were analysed by one-way ANOVA with Bonferroni post hoc
analysis. Chi-square test was used to analyse the composition of
aneurysm, the elastin degradation, and the incidence of aneurysm.
Survival rate was noted in Kaplan-Meier analysis and comparisons were
performed using the log-rank test. For angiotensin II-induced aortic
aneurysm, data analysis (survival rate, incidence, elastin degradation,
number of aneurysms) was performed on 11 mice in each group. In the
characteristic analysis of aneurysms (diameter, area, length, collagen
deposition), mice died of ruptured aneurysms (4 SKI mice treated with
solvent control) and mice without aneurysms (3 WT mice treated with
solvent control and 2 SKI mice treated with pioglitazone) were excluded.
For determining whether groups differ, the level of probability was set
at \emph{P} \textless{} .05 to constitute the threshold for statistical
significance.

\textbf{2.16 \textbar{} Materials}

\emph{Angiotensin II} was supplied by BACHEM (Cat\#
H-1705.0100).\emph{Pioglitazone} was supplied by MCE (Cat\# U72107).
PDTC was supplied by Macklin (Cat\# A800469). Buprenorphine was supplied
by Sigma-Aldrich (Cat\# BP1062). \emph{CsA} was supplied by MCE (Cat\#
HY-B0579).

\textbf{2.17 \textbar{} Nomenclature of targets and ligands}

Key protein targets and ligands in this article are hyperlinked to
corresponding entries in http://www.guidetopharmacology.org, the common
portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY
(\emph{RRID:SCR\_013077} ) (Harding et al., 2018), and are permanently
archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al.,
2019).

\textbf{3 \textbar{} Results}

\selectlanguage{greek}\textbf{3.1 \textbar{} Inactivation of SERCA2 C674 suppresses PPARγ by
activation of calcineurin/NFAT/NF-κB pathways}\selectlanguage{english}

Our previous study indicates that the inactivation of C674 by causing
the accumulation of intracellular Ca\textsuperscript{2+} to activate
calcineurin-mediated NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways, resulted in SMC phenotypic
modulation to accelerate aortic aneurysm. In order to study the
mechanism, we first performed RNA sequencing. Bioinformatics analysis
showed that inactivation of C674 had a wide range of effects on gene
expression, and a variety of signaling pathways were affected, among
which PPAR signaling pathway was the most closely related to the SMC
phenotypic modulation (Figure 1A). PPAR belongs to the nuclear receptor
superfamily, including PPAR\selectlanguage{greek}α, \selectlanguage{english}PPAR\selectlanguage{greek}β \selectlanguage{english}and PPAR\selectlanguage{greek}γ. \selectlanguage{english}FPKM values showed that
inactivation of C674 had no effect on \selectlanguage{greek}\emph{PPARα} \selectlanguage{english}(WT: 1.49 \selectlanguage{ngerman}± 0.28;
SKI: 1.06 ± 0.11) and \selectlanguage{greek}\emph{PPARβ} \selectlanguage{english}(WT: 25.13 \selectlanguage{ngerman}± 2.22; SKI: 29.74 ±
2.93), but decreased PPAR\selectlanguage{greek}γ\selectlanguage{english}2 (WT: 5.65 \selectlanguage{ngerman}± 0.68; SKI: 2.44 ± 0.21). The
down-regulation of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 was further confirmed at both mRNA level by
real-time quantitative PCR (Figure 1B) and protein level by Western blot
(Figure 1C) in aorta of SKI mice.

Activation of calcineurin or NF-\selectlanguage{greek}κ\selectlanguage{english}B inhibited the expression of PPAR\selectlanguage{greek}γ \selectlanguage{english}in
adipocytes (Liu et al., 2007) and pulmonary artery SMCs (Xie et al.,
2017). To explore whether C674 inactivation regulates the transcription
of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 by affecting its promoter activity, we constructed a mouse
PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter from -630 to +45 bp, containing NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B binding
sites. Compared with WT SMCs, the activity of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter in SKI
SMCs was down-regulated (Figure 1D), which could be reversed by either
calcineurin inhibitor CsA or NF-\selectlanguage{greek}κ\selectlanguage{english}B inhibitor PDTC (Figure 1E).
Correspondingly, compared with WT SMCs, the protein expression of PPAR\selectlanguage{greek}γ
\selectlanguage{english}was down-regulated in SKI SMCs, which could be reversed by either CsA
(Figure 1F) or PDTC (Figure 2A), suggesting that PPAR\selectlanguage{greek}γ \selectlanguage{english}might be the
downstream target of calcineurin/NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways. We have reported
that inactivation of C674 decreased the expression of MYOCD, increased
the expression of OPN, MMP2, Col I, Col III, phosphorylated p65NF-\selectlanguage{greek}κ\selectlanguage{english}B,
VCAM1, and ICAM1, accelerated SMC proliferation, migration and
macrophage adhesion to SMCs, and all these characters could be reversed
by CsA (Que et al., 2020). CsA inhibits the nuclear translocation of
NFAT4 and NF-\selectlanguage{greek}κ\selectlanguage{english}B in SKI SMCs (Que et al., 2020). Here we showed that
NF-\selectlanguage{greek}κ\selectlanguage{english}B inhibitor PDTC had similar effect to CsA in SKI SMCs (Figure 2),
further supporting that the activation of NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway caused by
inactivation of C674 promoted SMC phenotypic modulation. These results
indicate that inactivation of C674 promotes the binding of NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B to
PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter by increasing the nuclear translocation of NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B,
thus negatively regulating the transcription of PPAR\selectlanguage{greek}γ\selectlanguage{english}2, resulting in the
decrease of PPAR\selectlanguage{greek}γ \selectlanguage{english}protein expression.

\selectlanguage{greek}\textbf{3.2 \textbar{} The down-regulated PPARγ2accounts for the
phenotypic modulation of SKI SMCs}\selectlanguage{english}

\selectlanguage{greek}\emph{PPARγ} \selectlanguage{english}prevents the dedifferentiation of SMCs, restricts SMC
proliferation, migration and the activation of inflammatory pathway (Abe
et al., 2003; Hamblin et al., 2009). The two isoforms of PPAR\selectlanguage{greek}γ, \selectlanguage{english}PPAR\selectlanguage{greek}γ\selectlanguage{english}1
and PPAR\selectlanguage{greek}γ\selectlanguage{english}2, differ at their N terminal. The protective effect of PPAR\selectlanguage{greek}γ\selectlanguage{english}1
on the phenotypic modulation of SMCs was studied (Halabi et al., 2008;
Hu et al., 2008; Meredith et al., 2009). However, the role of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 in
the regulation of SMC phenotype is still unclear. Although PPAR\selectlanguage{greek}γ\selectlanguage{english}2 has 30
more amino acids than PPAR\selectlanguage{greek}γ\selectlanguage{english}1, it has similar DNA binding area and ligand
binding region to PPAR\selectlanguage{greek}γ\selectlanguage{english}1 (Werman et al., 1997). In SKI SMCs,
overexpression of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 increased the expression of MYOCD and decreased
the expression of OPN, MMP2, Col I, Col III, phosphorylated p65NF-\selectlanguage{greek}κ\selectlanguage{english}B,
VCAM1 and ICAM1 (Figure 3A), and inhibited cell proliferation,
migration, and macrophage adhesion to SMCs (Figure 3B-D). This provides
direct evidence that PPAR\selectlanguage{greek}γ\selectlanguage{english}2 inhibits the phenotypic modulation of SMCs,
and indicates that the decrease of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 is accountable for the
phenotypic modulation of SKI SMCs.

\selectlanguage{greek}\textbf{3.3 \textbar{} Activation of PPARγ prevents SMC phenotypic
modulation and represses the activities of NFAT/NF-κB in SKI SMCs}\selectlanguage{english}

Next, we used PPAR\selectlanguage{greek}γ \selectlanguage{english}activator pioglitazone to compensate for the
down-regulation of PPAR\selectlanguage{greek}γ \selectlanguage{english}expression in SKI SMCs. Activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}by
pioglitazone in SKI SMCs, similar to the overexpression of PPAR\selectlanguage{greek}γ\selectlanguage{english}2, also
increased the expression of MYOCD and decreased the expression of OPN,
MMP2, Col I, Col III, phosphorylated p65NF-\selectlanguage{greek}κ\selectlanguage{english}B, VCAM1 and ICAM1 (Figure
4A), and inhibited cell proliferation, migration, and macrophage
adhesion to SMCs (Figure 4B-D), which further confirmed the contribution
of the down-regulated PPAR\selectlanguage{greek}γ \selectlanguage{english}in promoting the phenotypic modulation of
SKI SMCs.

Though PPAR\selectlanguage{greek}γ \selectlanguage{english}could be a downstream target of calcineurin/NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B
signaling pathways, in return it could also interfere with NFAT and
NF-\selectlanguage{greek}κ\selectlanguage{english}B to repress their activities (Blanquart et al., 2003). Inactivation
of C674 increases the nuclear translocation of NFAT4 and NF-\selectlanguage{greek}κ\selectlanguage{english}B to be
active (Que et al., 2020). As shown in Figure 5, the activation of PPAR\selectlanguage{greek}γ
\selectlanguage{english}by pioglitazone could inhibit the nuclear translocation of NFAT4 and
NF-\selectlanguage{greek}κ\selectlanguage{english}B in SKI SMCs, suggesting the mutual negative regulation between
NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B and PPAR\selectlanguage{greek}γ \selectlanguage{english}in SMCs.

\selectlanguage{greek}\textbf{3.4 \textbar{} Activation of PPARγ by pioglitazone ameliorated
angiotensin II-induced aortic aneurysm in SKI mice}\selectlanguage{english}

Activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}by pioglitazone protected the formation and rupture
of experimental aortic aneurysms in mice (Golledge et al., 2010; Shimada
et al., 2015). Next, we used LDLR\textsuperscript{-/-} background mice
for angiotensin II-induced aortic aneurysm analysis, and pioglitazone or
solvent control administered 7 days after angiotensin II infusion
(Figure 6A). After 28 days of angiotensin II infusion, there was no
difference in body weight, plasma levels of triglyceride and cholesterol
among WT mice treated with solvent control, SKI mice treated with
solvent control, and SKI mice treated with pioglitazone (Table 1).

During 28-day angiotensin II infusion, all WT mice treated with solvent
control (11 out of 11) and SKI mice treated with pioglitazone (11 out of
11) survived, while 7 out of 11 SKI mice treated with solvent control
survived (Figure 6B). As shown in Figure 6C, 8 out of 11 WT mice treated
with solvent control developed aortic aneurysms. In contrast, all SKI
mice treated with solvent control (11 out of 11) developed aortic
aneurysms, in which 4 of them had early death due to the aneurysm
rupture. Pioglitazone slightly decreased the incidence of aortic
aneurysms in SKI mice (9 out of 11) comparable to WT mice treated with
solvent control. Compared with WT mice treated with solvent control, SKI
mice treated with solvent control had severer aortic aneurysm, of which
4 out of 11 had ruptured aneurysms. Pioglitazone ameliorated aortic
aneurysms in SKI mice with improved composition of aortic aneurysm
comparable to WT mice treated with solvent control (Figure 6D).
Accordingly, SKI mice treated with solvent control had a higher grade of
elastin degradation and a slight increase of collagen deposition than WT
mice treated with solvent control, and pioglitazone treatment could
reverse the increased elastin degradation and collagen deposition in SKI
mice comparable to WT mice treated with solvent control (Figure 6E\&F).

Mice (4 out of 11 SKI mice treated with solvent control) that died of
aneurysm rupture and mice without aneurysm (3 out of 11 WT mice treated
with solvent control and 2 out of 11 SKI mice treated with pioglitazone)
during the 28-day angiotensin II infusion were excluded from aneurysm
size analysis. The largest aneurysm in each mouse was analyzed. Compared
with WT mice treated with solvent control, in SKI mice treated with
solvent control, the diameter of aneurysm, the outer area of aneurysm,
and the media area with aneurysm, all showed a trend of increase, while
the lumen area showed a trend of decrease, and all these characters of
SKI mice could be reversed by pioglitazone treatment (Figure 7A). The
length and number of aneurysms were significantly larger in SKI mice
treated with solvent control compared with WT mice treated with solvent
control, while pioglitazone treatment reduced the length and number of
aneurysms in SKI mice (Figure 7B\&C).

In SKI non-aneurysmal aortas infused with angiotensin II, though
pioglitazone had no effect on the protein expression of PPAR\selectlanguage{greek}γ \selectlanguage{english}compared
with solvent control, it did increase the expression of MYOCD and
decrease the expression of OPN, MMP2, Col I, Col III, phosphorylated
p65NF-\selectlanguage{greek}κ\selectlanguage{english}B, VCAM1, and ICAM1 (Figure 8), further supporting the regulation
of PPAR\selectlanguage{greek}γ \selectlanguage{english}on these aneurysm-related proteins \emph{in vivo} . These
results indicated that inactivation of C674 by downregulation of PPAR\selectlanguage{greek}γ
\selectlanguage{english}to promote the phenotypic modulation of SMCs, thus aggravated
angiotensin II-induced aortic aneurysms, and pioglitazone by suppressing
these aneurysm-related proteins to improve aortic aneurysms.

\textbf{4 \textbar{} Discussion and conclusion}

SERCA2 is the main subtype of SERCA in the vascular system to maintain
calcium homeostasis. The C674 in the SERCA2 is evolutionarily conserved.
We previously reported that the irreversible oxidation of C674 occurred
broadly in mouse and human aortic aneurysms (Que et al., 2020). Using
SKI mice to mimic the persistent oxidative inactivation of C674, we find
that inactivation of C674 promotes the development of aortic aneurysm by
inducing SMC phenotypic modulation, which is caused by the accumulation
of intracellular Ca\textsuperscript{2+} that activates
Ca\textsuperscript{2+}-dependent calcineurin to promote the nuclear
translocation of NFAT and NF-\selectlanguage{greek}κ\selectlanguage{english}B to be active. In this study, we found
that the enhanced activation of NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways by C674 inactivation
suppressed the expression of PPAR\selectlanguage{greek}γ. \selectlanguage{english}In SKI mice, activation of PPAR\selectlanguage{greek}γ
\selectlanguage{english}blocked the activation of NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B, inhibited SMC phenotypic
modulation, and ameliorated angiotensin II-induced aortic aneurysms,
suggesting that the down-regulation of PPAR\selectlanguage{greek}γ \selectlanguage{english}by C674 inactivation is
critical to SMC phenotypic modulation and is responsible for the
accelerated aortic aneurysm in SKI mice. Our result indicates that the
redox state of C674 in the SERCA2 is pivotal in maintaining the balance
between calcineurin-mediated NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways and PPAR\selectlanguage{greek}γ. \selectlanguage{english}Inactivation
of C674 promotes the development of aortic aneurysm by inducing SMC
phenotypic modulation, which is caused by the imbalance between the
enhanced activation of calcineurin-mediated NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways and the
reduced expression of PPAR\selectlanguage{greek}γ.

\selectlanguage{english}PPAR\selectlanguage{greek}γ \selectlanguage{english}is a key downstream target of C674 inactivation, which triggers
the phenotypic modulation of SMCs and induces aortic aneurysm. PPAR\selectlanguage{greek}γ \selectlanguage{english}has
two major isoforms, PPAR\selectlanguage{greek}γ\selectlanguage{english}1 and PPAR\selectlanguage{greek}γ\selectlanguage{english}2, whose differ at N terminal. The
expression levels of PPAR\selectlanguage{greek}γ\selectlanguage{english}1 and PPAR\selectlanguage{greek}γ\selectlanguage{english}2 are similar in human SMCs (Limor
et al., 2008). PPAR\selectlanguage{greek}γ \selectlanguage{english}prevents the dedifferentiation of SMCs, restricts
SMC proliferation, migration and the activation of inflammatory pathways
(Hamblin et al., 2009). In vascular SMCs, dysregulation of PPAR\selectlanguage{greek}γ
\selectlanguage{english}upregulates protein expression of inflammatory factors, MMPs and OPN,
enhances cell proliferation and migration (Halabi et al., 2008; Hasan et
al., 2015; Meredith et al., 2009). Overexpression of PPAR\selectlanguage{greek}γ\selectlanguage{english}1 attenuates
atherosclerosis and stabilizes vulnerable plaques (Hu et al., 2008).
Lack of SMC PPAR\selectlanguage{greek}γ\selectlanguage{english}1 enhances SMC proliferation and migration, promotes
the vascular remodeling (Halabi et al., 2008; Meredith et al., 2009),
confirming the protective effect of PPAR\selectlanguage{greek}γ\selectlanguage{english}1 in SMC phenotypic modulation.
Rosiglitazone and pioglitazone, activators of both PPAR\selectlanguage{greek}γ\selectlanguage{english}1 and PPAR\selectlanguage{greek}γ\selectlanguage{english}2,
are beneficial in experimentally reducing aortic aneurysms (Blanquart et
al., 2003; Chen et al., 2015). The polymorphism of PPAR\selectlanguage{greek}γ\selectlanguage{english}2
(Pro\textsuperscript{12Ala}) is associated with a higher risk of
cardiovascular diseases, particularly myocardial infarction (Li et al.,
2015). However, it's not clear whether PPAR\selectlanguage{greek}γ\selectlanguage{english}2 contributes to SMC
phenotypic modulation and aortic aneurysm formation. The RNA sequencing
result indicated that PPAR\selectlanguage{greek}γ\selectlanguage{english}2 was down-regulated by C674 inactivation. In
SKI SMCs, overexpression of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 inhibited the expression of
aneurysm-related proteins and restricted SMC phenotypic modulation. Our
data provides a direct evidence of PPAR\selectlanguage{greek}γ\selectlanguage{english}2, similar to PPAR\selectlanguage{greek}γ\selectlanguage{english}1, in
restricting SMC phenotypic modulation. The reduced PPAR\selectlanguage{greek}γ\selectlanguage{english}2 is responsible
for SKI SMC phenotypic modulation. This highlights the critical role of
PPAR\selectlanguage{greek}γ \selectlanguage{english}regulated by the redox state of C674 in maintaining aortic
homeostasis.

NFAT and NF-\selectlanguage{greek}κ\selectlanguage{english}B share a number of properties, including similar DNA
binding domains and rapid nuclear translocation to response external
stimulus (Serfling et al., 2004). Activation of calcineurin or NF-\selectlanguage{greek}κ\selectlanguage{english}B
inhibits the expression of PPAR\selectlanguage{greek}γ \selectlanguage{english}in adipocytes (Liu et al., 2007) and
pulmonary artery SMCs (Xie et al., 2017). The regulation of NFAT or
NF-\selectlanguage{greek}κ\selectlanguage{english}B on PPAR\selectlanguage{greek}γ \selectlanguage{english}promoter activity hasn't been reported in aortic SMCs.
Here we showed that both the activity of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter and the protein
expression of PPAR\selectlanguage{greek}γ \selectlanguage{english}decrease in SKI SMCs, which are reversed by either
calcineurin inhibitor or NF-\selectlanguage{greek}κ\selectlanguage{english}B inhibitor, suggesting that PPAR\selectlanguage{greek}γ\selectlanguage{english}2 may be
a downstream target of calcineurin/NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling pathways and be
negatively regulated, in contrast to the positive regulation of PPAR\selectlanguage{greek}γ\selectlanguage{english}2
promoter activity by NFAT in human adipocytes (Kim et al., 2010) and
hepatoma cells (Yang et al., 2003). This diversity may be due to
different species, cell types, cofactors and other factors, such as
various subtypes and concentrations of NFAT.

Though PPAR\selectlanguage{greek}γ \selectlanguage{english}could be a downstream target of calcineurin/NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B
signaling pathways, in return it could also interfere with NFAT and
NF-\selectlanguage{greek}κ\selectlanguage{english}B (Blanquart et al., 2003). The activated PPAR\selectlanguage{greek}γ \selectlanguage{english}physically
associates with NFAT and blocks NFAT DNA binding and transcriptional
activity in T lymphocyte (Yang et al., 2000). In SMCs, PPAR\selectlanguage{greek}γ \selectlanguage{english}inhibits
NF-\selectlanguage{greek}κ\selectlanguage{english}B activity by promoting nuclear export of p65NF-\selectlanguage{greek}κ\selectlanguage{english}B, which is
abolished by dominant-negative mutation in PPAR\selectlanguage{greek}γ\selectlanguage{english}1 (Mukohda et al.,
2017). In SKI SMCs, overexpression of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 or activation of PPAR\selectlanguage{greek}γ
\selectlanguage{english}inhibits the expression of phosphorylated p65NF-\selectlanguage{greek}κ\selectlanguage{english}B, further confirmed
the mutual negative regulation between PPAR\selectlanguage{greek}γ \selectlanguage{english}and NF-\selectlanguage{greek}κ\selectlanguage{english}B. Activation of
PPAR\selectlanguage{greek}γ \selectlanguage{english}could inhibit the nuclear translocation of NFAT4 and NF-\selectlanguage{greek}κ\selectlanguage{english}B in SKI
SMCs. Although NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B and PPAR\selectlanguage{greek}γ \selectlanguage{english}can negatively regulate each other,
we infer that inactivation of C674 firstly activates intracellular
Ca\textsuperscript{2+}-dependent calcineurin/NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways to
suppress PPAR\selectlanguage{greek}γ \selectlanguage{english}due to its fundamental function in controlling the
concentration of intracellular Ca\textsuperscript{2+}. In return, the
down-regulated PPAR\selectlanguage{greek}γ \selectlanguage{english}further enhances the nuclear retention of
NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B, thus shifting the balance between NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B and PPAR\selectlanguage{greek}γ \selectlanguage{english}to
the continuous activation of NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B, consequently promoting SMC
phenotypic modulation and aortic aneurysm formation. Overexpression or
activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}could break this vicious circle and thus protect
from SMC phenotypic modulation and aortic aneurysm.

Angiotensin II is a common reagent for inducing aortic aneurysms in
rodents through complex mechanisms, including activation of calcineurin
and NFAT4 in arterial smooth muscle (Nieves-Cintron et al., 2007),
activation of NF-\selectlanguage{greek}κ\selectlanguage{english}B (Tsai et al., 2017), and down-regulation of PPAR\selectlanguage{greek}γ
\selectlanguage{english}(Subramanian et al., 2012). Activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}improves angiotensin
II-induced aortic aneurysm (Golledge et al., 2010; Motoki et al., 2015).
We have reported that angiotensin II could induce ROS production and
cause the irreversible oxidation of C674 in aorta (Que et al., 2020). As
data not shown, we found that angiotensin II downregulated PPAR\selectlanguage{greek}γ \selectlanguage{english}in
SMCs. Our study provided another mechanism for angiotensin II to
downregulate PPAR\selectlanguage{greek}γ, \selectlanguage{english}that is, to activate calcineurin/NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways
by causing irreversible oxidization of C674, thereby inhibiting PPAR\selectlanguage{greek}γ.
\selectlanguage{english}Inactivation of C674 in SKI mice might have a synergetic effect with
angiotensin II to aggravate aortic aneurysm.

Here, we used a relatively low dose of pioglitazone
(15 mg\selectlanguage{ngerman}·kg\selectlanguage{english}\textsuperscript{-1}*day\selectlanguage{english}\textsuperscript{-1}) instead of the
most commonly used dose
(50\selectlanguage{english} mg*kg\selectlanguage{english}\textsuperscript{-1}*day\selectlanguage{english}\textsuperscript{-1}) to activate
PPAR\selectlanguage{greek}γ\selectlanguage{english}\emph{in vivo} to minimize its effect on metabolism. Pioglitazone
is usually given before the infusion of angiotensin II to prevent the
formation of aortic aneurysm (Golledge et al., 2010). In this study,
pioglitazone was used 7 days after angiotensin II infusion to observe
the preventive and therapeutic effects of PPAR\selectlanguage{greek}γ \selectlanguage{english}activation on aortic
aneurysm. Our results indicate that early treatment with pioglitazone
can completely reverse the deterioration of aortic aneurysm in SKI mice,
similar to that in WT mice, suggesting that PPAR\selectlanguage{greek}γ \selectlanguage{english}activator or SERCA2
activator might be used in the treatment of early aortic aneurysm.
CDN1163 is a non-specific SERCA activator (Cornea et al., 2013), and its
effect on aortic aneurysm hasn't been reported. However, as the
structure of CDN1163 shows the potential of cytotoxicity, there is also
a need to develop other chemicals to selectively stimulate SERCA2. At
present, we are testing whether CDN1163 and other potential SERCA
activators can reverse the phenotypic modulation of SKI SMCs for further
application \emph{in vivo} .

In summary, our data provide direct evidence of the redox status of C674
in SERCA2 in maintaining aortic homeostasis by keeping the balance
between NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B and PPAR\selectlanguage{greek}γ. \selectlanguage{english}The irreversible oxidative inactivation of
SERCA2 C674 by increased levels of ROS under aortic aneurysm-prone
conditions, such as hypertension and aging, activates
Ca\textsuperscript{2+}-dependent calcineurin-mediated NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B
pathways that suppresses the expression of PPAR\selectlanguage{greek}γ, \selectlanguage{english}which in return causes
the sustained activation of NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B, leads to the phenotypic
modulation of SMCs, results in exacerbated aortic aneurysm.
Pharmacological activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}could restore the balance between
NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B and PPAR\selectlanguage{greek}γ \selectlanguage{english}to break this vicious circle, thus protecting
against SMC phenotypic modulation and aortic aneurysm caused by the
inactivation of SERCA2 C674. PPAR\selectlanguage{greek}γ \selectlanguage{english}and SERCA2 may be potential
therapeutic targets for aortic aneurysm.

\textbf{Acknowledgements}

The plasmids of pLG4.19, pRL-TK renilla, PPAR\selectlanguage{greek}γ\selectlanguage{english}2 and CMV-GFP were kindly
provided by Dr. Zhongzhou Yang and Dr. Xiaoqiu Xiao. This work was
supported by National Natural Science Foundation of China (31571172,
81870343, X.T., and 81700237, P.H.), Chongqing Research Program of Basic
Research and Frontier Technology (cstc2016jcyjA0407, X.T.), Fundamental
Research Funds for the Central Universities (2018CDQYYX0042, X.T. and
2018CDYXYX0027, P.H.).

\textbf{Author contributions}

Q., X.S., P. H. and X.T. participated in the research design and
performed the data analysis and interpretation; Y.Q. and X.T. wrote the
manuscript; Y.Q., X.S., L.W., and S.W. conducted the experiments; All
authors reviewed and revised the final version of this manuscript and
approved its submission.

\textbf{Conflict of interest}

The authors declare no conflicts of interest.

\textbf{Declaration of transparency and scientific rigor}

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



\textbf{Figure legends}

\textbf{FIGURE 1} Inactivation of SERCA2 C674 suppresses PPAR\selectlanguage{greek}γ \selectlanguage{english}by
activation of calcineurin/NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B pathways. (a) The main signaling
pathways from the Kyoto encyclopedia of genes and genomes pathway
enrichment analysis in duplicate aorta samples of WT and SKI mice in
LDLR\textsuperscript{-/-}background. (b) The mRNA levels of PPAR\selectlanguage{greek}γ\selectlanguage{english}2 in
aortas of WT and SKI mice in LDLR\textsuperscript{-/-} background. (c)
Representative Western blots of PPAR\selectlanguage{greek}γ \selectlanguage{english}from the aorta of WT and SKI mice
in LDLR\textsuperscript{-/-} background, and quantification of band
intensities in graph. (d) The mouse PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter activity in aortic
SMCs. (e) Effects of CsA or PDTC on PPAR\selectlanguage{greek}γ\selectlanguage{english}2 promoter activity in SKI
aortic SMCs. (f) Representative Western blots of PPAR\selectlanguage{greek}γ \selectlanguage{english}from aortic SMCs
and the quantification of band intensities in graph. (b)-(f), mean \selectlanguage{ngerman}±
SEM, unpaired Student's \emph{t} test, *\emph{P} \textless{} .05,
significantly different as indicated, \emph{n} = 5

\textbf{FIGURE 2} Activation of NF-\selectlanguage{greek}κ\selectlanguage{english}B accounts for the phenotypic
modulation of SKI aortic SMCs. (a) Representative Western blots from SKI
aortic SMCs treated with PDTC and quantification of band intensities in
graph. Mean \selectlanguage{ngerman}± SEM, unpaired Student's \emph{t} test,
*\emph{P}\textless{} .05, significantly different as indicated, \emph{n}
= 5. (b) Cell proliferation. (c) Cell migration. (d) Macrophage adhesion
to aortic SMCs. (b)-(d), mean ± SEM, unpaired Student's \emph{t} test,
*\emph{P} \textless{} .05, significantly different as indicated,\emph{n}
= 6

\textbf{FIGURE 3} The down-regulated PPAR\selectlanguage{greek}γ\selectlanguage{english}2 accounts for the phenotypic
modulation of SKI SMCs. (a) Representative Western blots from SKI aortic
SMCs transfected with PPAR\selectlanguage{greek}γ\selectlanguage{english}2 plasmids and quantification of band
intensities in graph. Mean \selectlanguage{ngerman}± SEM, unpaired Student's \emph{t} test,
*\emph{P} \textless{} .05, significantly different as indicated,\emph{n}
= 5. (b) Cell proliferation. (c) Cell migration. (d) Macrophage adhesion
to aortic SMCs. (b)-(d), mean ± SEM, unpaired Student's\emph{t} test,
*\emph{P} \textless{} .05, significantly different as indicated,
\emph{n} = 6

\textbf{FIGURE 4} Activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}by pioglitazone prevents the
phenotypic modulation of SKI SMCs. (a) Representative Western blots from
SKI aortic SMCs treated with pioglitazone and quantification of band
intensities in graph. Ctrl, solvent control; PIOG, pioglitazone. Mean \selectlanguage{ngerman}±
SEM, unpaired Student's \emph{t} test, *\emph{P} \textless{} .05,
significantly different as indicated, \emph{n} = 5. (b) Cell
proliferation. (c) Cell migration. (d) Macrophage adhesion to aortic
SMCs. (b)-(d), mean ± SEM, unpaired Student's \emph{t} test,
*\emph{P}\textless{} .05, significantly different as indicated, \emph{n}
= 6

\textbf{FIGURE 5} Activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}represses the activation of
NFAT/NF-\selectlanguage{greek}κ\selectlanguage{english}B in SKI aortic SMCs. Representative Western blots of NFAT4 and
p65NF-\selectlanguage{greek}κ\selectlanguage{english}B from SKI aortic SMCs treated with pioglitazone and
quantification of band intensities in graph. Histone H3 is the load
control of nuclear protein and GAPDH is the load control of total
protein. Ctrl, solvent control; PIOG, pioglitazone. Mean \selectlanguage{ngerman}± SEM, unpaired
Student's \emph{t} test, *\emph{P} \textless{} .05, significantly
different as indicated, \emph{n} = 5

\textbf{FIGURE 6} Activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}ameliorates angiotensin II-induced
aortic aneurysm in SKI mice. (a) The flow chart of animal procedure in
LDLR\textsuperscript{-/-} background mice. (b) Survival rate. SKI mice
treated with solvent control (SKI/Ctrl) vs. WT mice treated with solvent
control (WT/Ctrl) or SKI mice treated with pioglitazone (SKI/PIOG),
*\emph{P} \textless{} .05, Kaplan-Meier analysis and comparisons are
performed using the log-rank test. (c) Incidence. (d) Representative
photographs of aortic aneurysms and the summary of their composition in
graph. A red arrow indicates the location of the aneurysm. Ruptured
aneurysm, SKI/Ctrl vs. WT/Ctrl or SKI/PIOG, \emph{*P} \textless{} .05,
Chi-square test. (b)-(d), \emph{n} = 11. (e) Representative photographs
of elastin degradation and the percentage of different grades of elastin
degradation summarized in graph. Grade IV, SKI/Ctrl vs. WT/Ctrl or
SKI/PIOG, *\emph{P} \textless{} .05, Chi-square test. (f) Representative
photographs of collagen deposition and its score summary in graph.
(e)-(f), scale = 500 \selectlanguage{greek}μ\selectlanguage{english}m. Mean \selectlanguage{ngerman}± SEM, WT/Ctrl, \emph{n} = 8;
SKI/Ctrl,\emph{n} = 7; SKI/PIOG, \emph{n} = 9

\textbf{FIGURE 7} Activation of PPAR\selectlanguage{greek}γ \selectlanguage{english}decreased the size and number of
angiotensin II-induced aortic aneurysm in SKI mice. (a) Representative
photographs of HE staining of aortic aneurysms and the diameter and area
of aneurysm summarized in graph. Scale = 500 \selectlanguage{greek}μ\selectlanguage{english}m. (b) The length of
aneurysm. (c) The average number of aortic aneurysms in each group. Mean
\selectlanguage{ngerman}± SEM, One-way ANOVA, *\emph{P} \textless{} .05, significantly different
as indicated, WT/Ctrl, \emph{n} = 8; SKI/Ctrl, \emph{n} = 7;
SKI/PIOG,\emph{n} = 9

\textbf{FIGURE 8} Pioglitazone therapy inhibited the expression of
aneurysm related proteins in angiotensin II-infused SKI aorta. (a)
Representative Western blots from the aorta of SKI mice in
LDLR\selectlanguage{english}\textsuperscript{-/-} background infused with angiotensin II, and
quantification of band intensities in graph. Ang II, angiotensin II;
Ctrl, solvent control; PIOG, pioglitazone. Mean +- SEM, unpaired
Student's \emph{t} test, *\emph{P} \textless{} .05, significantly
different as indicated, \emph{n} = 5

\textbf{REFERENCES}

Abe M, Hasegawa K, Wada H, Morimoto T, Yanazume T, Kawamura T, . . .
Kita T (2003). GATA-6 is involved in PPARgamma-mediated activation of
differentiated phenotype in human vascular smooth muscle cells.
Arterioscler Thromb Vasc Biol 23\textbf{:} 404-410.

Adachi T, Weisbrod RM, Pimentel DR, Ying J, Sharov VS, Schoneich C, \&
Cohen RA (2004). S-Glutathiolation by peroxynitrite activates SERCA
during arterial relaxation by nitric oxide. Nat Med
10\textbf{:}1200-1207.

Ailawadi G, Moehle CW, Pei H, Walton SP, Yang Z, Kron IL, . . . Owens GK
(2009). Smooth muscle phenotypic modulation is an early event in aortic
aneurysms. J Thorac Cardiovasc Surg 138\textbf{:} 1392-1399.

Alexander SPH, Kelly E, Mathie A, Peters JA, Veale EL, Armstrong JF, . .
. Collaborators C (2019). THE CONCISE GUIDE TO PHARMACOLOGY 2019/20:
Introduction and Other Protein Targets. Br J Pharmacol 176 Suppl
1\textbf{:} S1-S20.

Bhamidipati CM, Whatling CA, Mehta GS, Meher AK, Hajzus VA, Su G, . . .
Ailawadi G (2014). 5-Lipoxygenase pathway in experimental abdominal
aortic aneurysms. Arterioscler Thromb Vasc Biol 34\textbf{:} 2669-2678.

Blanquart C, Barbier O, Fruchart JC, Staels B, \& Glineur C (2003).
Peroxisome proliferator-activated receptors: regulation of
transcriptional activities and roles in inflammation. J Steroid Biochem
Mol Biol 85\textbf{:} 267-273.

Chen R, Yan J, Liu P, \& Wang Z (2015). Effects of thiazolidinedione
therapy on inflammatory markers of type 2 diabetes: a meta-analysis of
randomized controlled trials. PLoS One 10\textbf{:} e0123703.

Cornea RL, Gruber SJ, Lockamy EL, Muretta JM, Jin D, Chen J, . . .
Thomas DD (2013). High-throughput FRET assay yields allosteric SERCA
activators. Journal of biomolecular screening 18\textbf{:} 97-107.

Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SP, Giembycz MA, . .
. McGrath JC (2015). Experimental design and analysis and their
reporting: new guidance for publication in BJP. Br J Pharmacol
172\textbf{:} 3461-3471.

de Winther MP, Kanters E, Kraal G, \& Hofker MH (2005). Nuclear factor
kappaB signaling in atherogenesis. Arterioscler Thromb Vasc Biol
25\textbf{:} 904-914.

Golledge J, Cullen B, Rush C, Moran CS, Secomb E, Wood F, . . . Norman
PE (2010). Peroxisome proliferator-activated receptor ligands reduce
aortic dilatation in a mouse model of aortic aneurysm. Atherosclerosis
210\textbf{:} 51-56.

Golledge J, Muller J, Shephard N, Clancy P, Smallwood L, Moran C, . . .
Norman PE (2007). Association between osteopontin and human abdominal
aortic aneurysm. Arterioscler Thromb Vasc Biol 27\textbf{:} 655-660.

Halabi CM, Beyer AM, de Lange WJ, Keen HL, Baumbach GL, Faraci FM, \&
Sigmund CD (2008). Interference with PPAR gamma function in smooth
muscle causes vascular dysfunction and hypertension. Cell Metab
7\textbf{:} 215-226.

Hamblin M, Chang L, Fan Y, Zhang J, \& and Chen Y (2009). PPARs and the
cardiovascular system. Antioxid Redox Signal 11\textbf{:} 1415-1452.

Harding SD, Sharman JL, Faccenda E, Southan C, Pawson AJ, Ireland S, . .
. Nc I (2018). The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: updates and
expansion to encompass the new guide to IMMUNOPHARMACOLOGY. Nucleic
Acids Res 46\textbf{:} D1091-D1106.

Hasan DM, Starke RM, Gu H, Wilson K, Chu Y, Chalouhi N, . . . Sigmund CD
(2015). Smooth Muscle Peroxisome Proliferator-Activated Receptor gamma
Plays a Critical Role in Formation and Rupture of Cerebral Aneurysms in
Mice In Vivo. Hypertension 66\textbf{:} 211-220.

Hu Q, Zhang XJ, Zhang C, Zhao YX, He H, Liu CX, . . . Zhang Y (2008).
Peroxisome proliferator-activated receptor-gamma1 gene therapy
attenuates atherosclerosis and stabilizes plaques in apolipoprotein
E-deficient mice. Hum Gene Ther 19\textbf{:} 287-299.

Kim HB, Kumar A, Wang L, Liu GH, Keller SR, Lawrence JC, Jr., . . .
Harris TE (2010). Lipin 1 represses NFATc4 transcriptional activity in
adipocytes to inhibit secretion of inflammatory factors. Mol Cell Biol
30\textbf{:} 3126-3139.

Li Y, Zhu J, \& Ding JQ (2015). Association of the PPARgamma2 Pro12Ala
polymorphism with increased risk of cardiovascular diseases. Genet Mol
Res 14\textbf{:} 18662-18674.

Limor R, Sharon O, Knoll E, Many A, Weisinger G, \& Stern N (2008).
Lipoxygenase-derived metabolites are regulators of peroxisome
proliferator-activated receptor gamma-2 expression in human vascular
smooth muscle cells. Am J Hypertens 21\textbf{:} 219-223.

Liu G, Wu F, Jiang X, Que Y, Qin Z, Hu P, . . . Tong X (2020).
Inactivation of Cys(674) in SERCA2 increases BP by inducing endoplasmic
reticulum stress and soluble epoxide hydrolase. Br J Pharmacol
177\textbf{:} 1793-1805.

Liu L, \& Clipstone NA (2007). Prostaglandin F2alpha inhibits adipocyte
differentiation via a G alpha q-calcium-calcineurin-dependent signaling
pathway. J Cell Biochem 100\textbf{:} 161-173.

Meng YH, Tian C, Liu L, Wang L, \& Chang Q (2014). Elevated expression
of connective tissue growth factor, osteopontin and increased collagen
content in human ascending thoracic aortic aneurysms. Vascular
22\textbf{:} 20-27.

Meredith D, Panchatcharam M, Miriyala S, Tsai YS, Morris AJ, Maeda N, .
. . Smyth SS (2009). Dominant-negative loss of PPARgamma function
enhances smooth muscle cell proliferation, migration, and vascular
remodeling. Arterioscler Thromb Vasc Biol 29\textbf{:} 465-471.

Miyake T, Aoki M, Masaki H, Kawasaki T, Oishi M, Kataoka K, . . .
Morishita R (2007). Regression of abdominal aortic aneurysms by
simultaneous inhibition of nuclear factor kappaB and ets in a rabbit
model. Circ Res 101\textbf{:} 1175-1184.

Monaco C, \& Paleolog E (2004). Nuclear factor kappaB: a potential
therapeutic target in atherosclerosis and thrombosis. Cardiovasc Res
61\textbf{:} 671-682.

Motoki T, Kurobe H, Hirata Y, Nakayama T, Kinoshita H, Rocco KA, . . .
Kitagawa T (2015). PPAR-gamma agonist attenuates inflammation in aortic
aneurysm patients. Gen Thorac Cardiovasc Surg 63\textbf{:} 565-571.

Mukohda M, Lu KT, Guo DF, Wu J, Keen HL, Liu X, . . . Sigmund CD (2017).
Hypertension-Causing Mutation in Peroxisome Proliferator-Activated
Receptor gamma Impairs Nuclear Export of Nuclear Factor-kappaB p65 in
Vascular Smooth Muscle. Hypertension 70\textbf{:} 174-182.

Nakao T, Horie T, Baba O, Nishiga M, Nishino T, Izuhara M, . . . Ono K
(2017). Genetic Ablation of MicroRNA-33 Attenuates Inflammation and
Abdominal Aortic Aneurysm Formation via Several Anti-Inflammatory
Pathways. Arterioscler Thromb Vasc Biol 37\textbf{:} 2161-2170.

Nieves-Cintron M, Amberg GC, Nichols CB, Molkentin JD, \& Santana LF
(2007). Activation of NFATc3 down-regulates the beta1 subunit of large
conductance, calcium-activated K+ channels in arterial smooth muscle and
contributes to hypertension. J Biol Chem 282\textbf{:} 3231-3240.

Qin F, Siwik DA, Lancel S, Zhang J, Kuster GM, Luptak I, . . . Colucci
WS (2013). Hydrogen peroxide-mediated SERCA cysteine 674 oxidation
contributes to impaired cardiac myocyte relaxation in senescent mouse
heart. J Am Heart Assoc 2\textbf{:} e000184.

Que Y, Shu X, Wang L, Hu P, Wang S, Xiong R, . . . Tong X (2020).
Inactivation of cysteine 674 in the SERCA2 accelerates experimental
aortic aneurysm. J Mol Cell Cardiol.

Serfling E, Berberich-Siebelt F, Avots A, Chuvpilo S, Klein-Hessling S,
Jha MK, . . . Palmetshofer A (2004). NFAT and NF-kappaB factors-the
distant relatives. Int J Biochem Cell Biol 36\textbf{:} 1166-1170.

Shimada K, Furukawa H, Wada K, Korai M, Wei Y, Tada Y, . . . Hashimoto T
(2015). Protective Role of Peroxisome Proliferator-Activated
Receptor-gamma in the Development of Intracranial Aneurysm Rupture.
Stroke 46\textbf{:} 1664-1672.

Subramanian V, Golledge J, Heywood EB, Bruemmer D, \& Daugherty A
(2012). Regulation of peroxisome proliferator-activated receptor-gamma
by angiotensin II via transforming growth factor-beta1-activated p38
mitogen-activated protein kinase in aortic smooth muscle cells.
Arterioscler Thromb Vasc Biol 32\textbf{:} 397-405.

Tang RH, Zheng XL, Callis TE, Stansfield WE, He J, Baldwin AS, . . .
Selzman CH (2008). Myocardin inhibits cellular proliferation by
inhibiting NF-kappaB(p65)-dependent cell cycle progression. Proc Natl
Acad Sci U S A 105\textbf{:} 3362-3367.

Thompson MD, Mei Y, Weisbrod RM, Silver M, Shukla PC, Bolotina VM, . . .
Tong X (2014). Glutathione adducts on sarcoplasmic/endoplasmic reticulum
Ca2+ ATPase Cys-674 regulate endothelial cell calcium stores and
angiogenic function as well as promote ischemic blood flow recovery. J
Biol Chem 289\textbf{:} 19907-19916.

Tong X, Khandelwal AR, Qin Z, Wu X, Chen L, Ago T, . . . Cohen RA
(2015). Role of smooth muscle Nox4-based NADPH oxidase in neointimal
hyperplasia. J Mol Cell Cardiol 89\textbf{:} 185-194.

Tong X, Khandelwal AR, Wu X, Xu Z, Yu W, Chen C, . . . Zeng C (2016).
Pro-atherogenic role of smooth muscle Nox4-based NADPH oxidase. J Mol
Cell Cardiol 92\textbf{:} 30-40.

Tong X, Ying J, Pimentel DR, Trucillo M, Adachi T, \& Cohen RA (2008).
High glucose oxidizes SERCA cysteine-674 and prevents inhibition by
nitric oxide of smooth muscle cell migration. J Mol Cell Cardiol
44\textbf{:} 361-369.

Tsai MH, Lee CW, Hsu LF, Li SY, Chiang YC, Lee MH, . . . Lee IT (2017).
CO-releasing molecules CORM2 attenuates angiotensin II-induced human
aortic smooth muscle cell migration through inhibition of ROS/IL-6
generation and matrix metalloproteinases-9 expression. Redox Biol
12\textbf{:} 377-388.

Wang L, Zhang J, Fu W, Guo D, Jiang J, \& Wang Y (2012a). Association of
smooth muscle cell phenotypes with extracellular matrix disorders in
thoracic aortic dissection. J Vasc Surg 56\textbf{:} 1698-1709, 1709
e1691.

Wang S, Zhang C, Zhang M, Liang B, Zhu H, Lee J, . . . Zou MH (2012b).
Activation of AMP-activated protein kinase alpha2 by nicotine instigates
formation of abdominal aortic aneurysms in mice in vivo. Nat Med
18\textbf{:} 902-910.

Werman A, Hollenberg A, Solanes G, Bjorbaek C, Vidal-Puig AJ, \& Flier
JS (1997). Ligand-independent activation domain in the N terminus of
peroxisome proliferator-activated receptor gamma (PPARgamma).
Differential activity of PPARgamma1 and -2 isoforms and influence of
insulin. J Biol Chem 272\textbf{:} 20230-20235.

Xie X, Li S, Zhu Y, Liu L, Pan Y, Wang J, . . . Li M (2017).
MicroRNA-27a/b mediates endothelin-1-induced PPARgamma reduction and
proliferation of pulmonary artery smooth muscle cells. Cell Tissue Res
369\textbf{:} 527-539.

Yang TT, \& Chow CW (2003). Transcription cooperation by NFAT.C/EBP
composite enhancer complex. J Biol Chem 278\textbf{:} 15874-15885.

Yang XY, Wang LH, Chen T, Hodge DR, Resau JH, DaSilva L, \& Farrar WL
(2000). Activation of human T lymphocytes is inhibited by peroxisome
proliferator-activated receptor gamma (PPARgamma) agonists. PPARgamma
co-association with transcription factor NFAT. J Biol Chem
275\textbf{:}4541-4544.

Ying J, Sharov V, Xu S, Jiang B, Gerrity R, Schoneich C, \& Cohen RA
(2008). Cysteine-674 oxidation and degradation of sarcoplasmic reticulum
Ca(2+) ATPase in diabetic pig aorta. Free Radic Biol Med
45\textbf{:}756-762.

\textbf{Hosted file}

\verb`SKI-AA-BJP-Table.docx`  available at \url{https://authorea.com/users/353973/articles/477687-inactivation-of-serca2-cys674-accelerates-aortic-aneurysms-by-suppressing-ppar%CE%B3}

\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/AA-BJP/AA-BJP}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

