4 | 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 Ca2+ that activates Ca2+-dependent calcineurin to promote the nuclear translocation of NFAT and NF-κB to be active. In this study, we found that the enhanced activation of NFAT/NF-κB pathways by C674 inactivation suppressed the expression of PPARγ. In SKI mice, activation of PPARγ blocked the activation of NFAT/NF-κB, inhibited SMC phenotypic modulation, and ameliorated angiotensin II-induced aortic aneurysms, suggesting that the down-regulation of PPARγ 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-κB pathways and PPARγ. 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-κB pathways and the reduced expression of PPARγ.
PPARγ is a key downstream target of C674 inactivation, which triggers the phenotypic modulation of SMCs and induces aortic aneurysm. PPARγ has two major isoforms, PPARγ1 and PPARγ2, whose differ at N terminal. The expression levels of PPARγ1 and PPARγ2 are similar in human SMCs (Limor et al., 2008). PPARγ prevents the dedifferentiation of SMCs, restricts SMC proliferation, migration and the activation of inflammatory pathways (Hamblin et al., 2009). In vascular SMCs, dysregulation of PPARγ 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 PPARγ1 attenuates atherosclerosis and stabilizes vulnerable plaques (Hu et al., 2008). Lack of SMC PPARγ1 enhances SMC proliferation and migration, promotes the vascular remodeling (Halabi et al., 2008; Meredith et al., 2009), confirming the protective effect of PPARγ1 in SMC phenotypic modulation. Rosiglitazone and pioglitazone, activators of both PPARγ1 and PPARγ2, are beneficial in experimentally reducing aortic aneurysms (Blanquart et al., 2003; Chen et al., 2015). The polymorphism of PPARγ2 (Pro12Ala) is associated with a higher risk of cardiovascular diseases, particularly myocardial infarction (Li et al., 2015). However, it’s not clear whether PPARγ2 contributes to SMC phenotypic modulation and aortic aneurysm formation. The RNA sequencing result indicated that PPARγ2 was down-regulated by C674 inactivation. In SKI SMCs, overexpression of PPARγ2 inhibited the expression of aneurysm-related proteins and restricted SMC phenotypic modulation. Our data provides a direct evidence of PPARγ2, similar to PPARγ1, in restricting SMC phenotypic modulation. The reduced PPARγ2 is responsible for SKI SMC phenotypic modulation. This highlights the critical role of PPARγ regulated by the redox state of C674 in maintaining aortic homeostasis.
NFAT and NF-κB 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-κB inhibits the expression of PPARγ in adipocytes (Liu et al., 2007) and pulmonary artery SMCs (Xie et al., 2017). The regulation of NFAT or NF-κB on PPARγ promoter activity hasn’t been reported in aortic SMCs. Here we showed that both the activity of PPARγ2 promoter and the protein expression of PPARγ decrease in SKI SMCs, which are reversed by either calcineurin inhibitor or NF-κB inhibitor, suggesting that PPARγ2 may be a downstream target of calcineurin/NFAT/NF-κB signaling pathways and be negatively regulated, in contrast to the positive regulation of PPARγ2 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 PPARγ could be a downstream target of calcineurin/NFAT/NF-κB signaling pathways, in return it could also interfere with NFAT and NF-κB (Blanquart et al., 2003). The activated PPARγ physically associates with NFAT and blocks NFAT DNA binding and transcriptional activity in T lymphocyte (Yang et al., 2000). In SMCs, PPARγ inhibits NF-κB activity by promoting nuclear export of p65NF-κB, which is abolished by dominant-negative mutation in PPARγ1 (Mukohda et al., 2017). In SKI SMCs, overexpression of PPARγ2 or activation of PPARγ inhibits the expression of phosphorylated p65NF-κB, further confirmed the mutual negative regulation between PPARγ and NF-κB. Activation of PPARγ could inhibit the nuclear translocation of NFAT4 and NF-κB in SKI SMCs. Although NFAT/NF-κB and PPARγ can negatively regulate each other, we infer that inactivation of C674 firstly activates intracellular Ca2+-dependent calcineurin/NFAT/NF-κB pathways to suppress PPARγ due to its fundamental function in controlling the concentration of intracellular Ca2+. In return, the down-regulated PPARγ further enhances the nuclear retention of NFAT/NF-κB, thus shifting the balance between NFAT/NF-κB and PPARγ to the continuous activation of NFAT/NF-κB, consequently promoting SMC phenotypic modulation and aortic aneurysm formation. Overexpression or activation of PPARγ 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-κB (Tsai et al., 2017), and down-regulation of PPARγ (Subramanian et al., 2012). Activation of PPARγ 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 PPARγ in SMCs. Our study provided another mechanism for angiotensin II to downregulate PPARγ, that is, to activate calcineurin/NFAT/NF-κB pathways by causing irreversible oxidization of C674, thereby inhibiting PPARγ. 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·kg−1·day−1) instead of the most commonly used dose (50 mg·kg−1·day−1) to activate PPARγin 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 PPARγ 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 PPARγ 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 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-κB and PPARγ. The irreversible oxidative inactivation of SERCA2 C674 by increased levels of ROS under aortic aneurysm-prone conditions, such as hypertension and aging, activates Ca2+-dependent calcineurin-mediated NFAT/NF-κB pathways that suppresses the expression of PPARγ, which in return causes the sustained activation of NFAT/NF-κB, leads to the phenotypic modulation of SMCs, results in exacerbated aortic aneurysm. Pharmacological activation of PPARγ could restore the balance between NFAT/NF-κB and PPARγ to break this vicious circle, thus protecting against SMC phenotypic modulation and aortic aneurysm caused by the inactivation of SERCA2 C674. PPARγ and SERCA2 may be potential therapeutic targets for aortic aneurysm.