3.1 | Inactivation of SERCA2 C674 suppresses PPARγ by
activation of calcineurin/NFAT/NF-κB pathways
Our previous study indicates that the inactivation of C674 by causing
the accumulation of intracellular Ca2+ to activate
calcineurin-mediated NFAT/NF-κB 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 PPARα, PPARβ and PPARγ. FPKM values showed that
inactivation of C674 had no effect on PPARα (WT: 1.49 ± 0.28;
SKI: 1.06 ± 0.11) and PPARβ (WT: 25.13 ± 2.22; SKI: 29.74 ±
2.93), but decreased PPARγ2 (WT: 5.65 ± 0.68; SKI: 2.44 ± 0.21). The
down-regulation of PPARγ2 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-κB inhibited the expression of PPARγ in
adipocytes (Liu et al., 2007) and pulmonary artery SMCs (Xie et al.,
2017). To explore whether C674 inactivation regulates the transcription
of PPARγ2 by affecting its promoter activity, we constructed a mouse
PPARγ2 promoter from -630 to +45 bp, containing NFAT/NF-κB binding
sites. Compared with WT SMCs, the activity of PPARγ2 promoter in SKI
SMCs was down-regulated (Figure 1D), which could be reversed by either
calcineurin inhibitor CsA or NF-κB inhibitor PDTC (Figure 1E).
Correspondingly, compared with WT SMCs, the protein expression of PPARγ
was down-regulated in SKI SMCs, which could be reversed by either CsA
(Figure 1F) or PDTC (Figure 2A), suggesting that PPARγ might be the
downstream target of calcineurin/NFAT/NF-κB 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-κB,
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-κB in SKI SMCs (Que et al., 2020). Here we showed that
NF-κB inhibitor PDTC had similar effect to CsA in SKI SMCs (Figure 2),
further supporting that the activation of NF-κB pathway caused by
inactivation of C674 promoted SMC phenotypic modulation. These results
indicate that inactivation of C674 promotes the binding of NFAT/NF-κB to
PPARγ2 promoter by increasing the nuclear translocation of NFAT/NF-κB,
thus negatively regulating the transcription of PPARγ2, resulting in the
decrease of PPARγ protein expression.
3.2 | The down-regulated PPARγ2accounts for the
phenotypic modulation of SKI SMCs
PPARγ 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 PPARγ, PPARγ1
and PPARγ2, differ at their N terminal. The protective effect of PPARγ1
on the phenotypic modulation of SMCs was studied (Halabi et al., 2008;
Hu et al., 2008; Meredith et al., 2009). However, the role of PPARγ2 in
the regulation of SMC phenotype is still unclear. Although PPARγ2 has 30
more amino acids than PPARγ1, it has similar DNA binding area and ligand
binding region to PPARγ1 (Werman et al., 1997). In SKI SMCs,
overexpression of PPARγ2 increased the expression of MYOCD and decreased
the expression of OPN, MMP2, Col I, Col III, phosphorylated p65NF-κB,
VCAM1 and ICAM1 (Figure 3A), and inhibited cell proliferation,
migration, and macrophage adhesion to SMCs (Figure 3B-D). This provides
direct evidence that PPARγ2 inhibits the phenotypic modulation of SMCs,
and indicates that the decrease of PPARγ2 is accountable for the
phenotypic modulation of SKI SMCs.
3.3 | Activation
of PPARγ prevents SMC phenotypic modulation and represses the activities
of NFAT/NF-κB in SKI SMCs
Next, we used PPARγ activator pioglitazone to compensate for the
down-regulation of PPARγ expression in SKI SMCs. Activation of PPARγ by
pioglitazone in SKI SMCs, similar to the overexpression of PPARγ2, also
increased the expression of MYOCD and decreased the expression of OPN,
MMP2, Col I, Col III, phosphorylated p65NF-κB, 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 PPARγ in promoting the phenotypic modulation of
SKI SMCs.
Though PPARγ could be a downstream target of calcineurin/NFAT/NF-κB
signaling pathways, in return it could also interfere with NFAT and
NF-κB to repress their activities (Blanquart et al., 2003). Inactivation
of C674 increases the nuclear translocation of NFAT4 and NF-κB to be
active (Que et al., 2020). As shown in Figure 5, the activation of PPARγ
by pioglitazone could inhibit the nuclear translocation of NFAT4 and
NF-κB in SKI SMCs, suggesting the mutual negative regulation between
NFAT/NF-κB and PPARγ in SMCs.
3.4 | Activation
of PPARγ by pioglitazone ameliorated angiotensin II-induced aortic
aneurysm in SKI mice
Activation of PPARγ 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−/− 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 PPARγ compared
with solvent control, it did increase the expression of MYOCD and
decrease the expression of OPN, MMP2, Col I, Col III, phosphorylated
p65NF-κB, VCAM1, and ICAM1 (Figure 8), further supporting the regulation
of PPARγ on these aneurysm-related proteins in vivo . These
results indicated that inactivation of C674 by downregulation of PPARγ
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.