Activation of the IKK2/NF-κB pathway in VSMCs inhibits calcified vascular stiffness in CKD

IKK2/NF-κB pathway–mediated inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2/NF-κB pathway in medial calcification remains to be elucidated. In this study, we found that chronic kidney disease (CKD) induces inflammatory pathways through the local activation of the IKK2/NF-κB pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2/NF-κB pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF-κB by SMC-specific IκBα deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2/NF-κB pathway induced cell death of VSMCs by reducing anti–cell death gene expression, whereas activation of NF-κB reduced CKD-dependent vascular cell death. In addition, increased calcification of extracellular vesicles through the inhibition of the IKK2/NF-κB pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2/NF-κB pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles

Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells

Emerging evidence indicates that upregulation of the ER stress–induced pro-osteogenic transcription factor ATF4 plays an important role in vascular calcification, a common complication in patients with aging, diabetes, and chronic kidney disease (CKD). In this study, we demonstrated the pathophysiological role of ATF4 in vascular calcification using global Atf4 KO, smooth muscle cell–specific (SMC-specific) Atf4 KO, and transgenic (TG) mouse models. Reduced expression of ATF4 in global ATF4-haplodeficient and SMC-specific Atf4 KO mice reduced medial and atherosclerotic calcification under normal kidney and CKD conditions. In contrast, increased expression of ATF4 in SMC-specific Atf4 TG mice caused severe medial and atherosclerotic calcification. We further demonstrated that ATF4 transcriptionally upregulates the expression of type III sodium-dependent phosphate cotransporters (PiT1 and PiT2) by interacting with C/EBPβ. These results demonstrate that the ER stress effector ATF4 plays a critical role in the pathogenesis of vascular calcification through increased phosphate uptake in vascular SMCs

Dual Activation of the Bile Acid Nuclear Receptor FXR and G-Protein-Coupled Receptor TGR5 Protects Mice against Atherosclerosis

<div><p>Bile acid signaling is a critical regulator of glucose and energy metabolism, mainly through the nuclear receptor FXR and the G protein-coupled receptor TGR. The purpose of the present study was to investigate whether dual activation of FXR and TGR5 plays a significant role in the prevention of atherosclerosis progression. To evaluate the effects of bile acid signaling in atherogenesis, ApoE^−/− mice and LDLR^−/− mice were treated with an FXR/TGR5 dual agonist (INT-767). INT-767 treatment drastically reduced serum cholesterol levels. INT-767 treatment significantly reduced atherosclerotic plaque formation in both ApoE^−/− and LDLR^−/− mice. INT-767 decreased the expression of pro-inflammatory cytokines and chemokines in the aortas of ApoE^−/− mice through the inactivation of NF-κB. In addition, J774 macrophages treated with INT-767 had significantly lower levels of active NF-κB, resulting in cytokine production in response to LPS through a PKA dependent mechanism. This study demonstrates that concurrent activation of FXR and TGR5 attenuates atherosclerosis by reducing both circulating lipids and inflammation.</p></div

INT-767 reduces high fat-induced adiposity and hyperlipidemia in ApoE^−/− mice.

<p>Mice were treated with INT-767 as indicated in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108270#s2" target="_blank">Methods</a></i> section. A) Body weight of ApoE^−/− mice treated with INT-767. B) White fat weight of ApoE^−/− mice. C) Cholesterol distribution in the lipoproteins of ApoE^−/− mice treated with INT-767. D) Triglyceride distribution in the lipoproteins of mice treated with INT-767. E) Body weight of LDLR^−/− mice treated with INT-767. F) Cholesterol distribution in the lipoproteins of LDLR^−/− mice treated with INT-767.</p

INT-767 inhibits the development of aortic lesions in ApoE^−/− and LDLR^−/− mice.

<p>Atherosclerotic lesions were quantified by en face analysis. A) Representative picture of en face analysis of atherosclerosis in ApoE^−/− mice treated with INT-767. B) Quantification of en face analysis in ApoE^−/− mice treated with INT-767 for 12 weeks. C) Representative picture of en face analysis of atherosclerosis in LDLR^−/− mice treated with INT-767. D) Quantification of en face analysis in LDLR^−/− mice treated with INT-767 for 16 weeks. ***P<0.001.</p

INT-767 reduces cholic acid and its metabolites but not chenodeoxycholic acid though the reduction of hepatic CYP7A1 and CYP8B1 expression in ApoE^−/− mice.

<p>A) mRNA levels of hepatic FXR targets in ApoE^−/− mice treated with INT-767. B) Serum bile acid content in ApoE^−/− mice treated with INT-767. CA, cholic acid; MCA, muricholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; T, tauro. *P<0.05 and ***P<0.001.</p

Serum lipid levels in ApoE^−/− mice treated with INT-767 for 12 weeks.

<p>Eight-week-old mice were fed a Western diet containing INT-767 for 12 weeks.</p><p>Blood was drawn after 4 hours of fasting.</p><p>Data expressed as Mean ± SEM.</p><p>*p<0.05 vs. ApoE^−/− mice with vehicle.</p><p>Serum lipid levels in ApoE^−/− mice treated with INT-767 for 12 weeks.</p

INT-767 inhibits activation of NF-κB and cytokine production through a TGR5-PKA-dependent mechanism.

<p>A) TNFα and B) IL-1β mRNA levels, C) NF-κB binding activity by EMSA and D) the densitometric analysis of the NF-κB EMSA. J774.2 macrophages were pretreated with 10 µM INT-767 for 2 hours and treated with LPS (100 ng/ml) for 2 hours in the presence of a PKA inhibitor (1 mM, PKAI, Rp-8-Br-cAMPS). E) TGR5 or F) FXR was overexpressed in Raw294.7 macrophages, which express very low levels of TGR5 and FXR. G) INT-767 inhibits TNFα expression induced by LPS (100 ng/ml) in Raw294.7 macrophages overexpressing TGR5 but not FXR. * P<0.05, **P<0.01, and ***P<0.001.</p

INT-767 reduces high fat-induced adiposity and hyperlipidemia in LDLR^−/− mice.

<p>Mice were treated with INT-767 as indicated in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108270#s2" target="_blank">Methods</a></i> section. A) Body weight of LDLR^−/− mice treated with INT-767. B) White fat weight of ApoE^−/− mice. C) Cholesterol distribution in the lipoproteins of LDLR^−/− mice treated with INT-767. D) Triglyceride distribution in the lipoproteins of mice treated with INT-767. **P<0.001 and ***P<0.001.</p

Serum lipid levels in LDLR^−/− mice treated with INT-767.

<p>Eight-week-old mice were fed a Western diet containing INT-767 for 16 weeks.</p><p>Blood was drawn after 4 hours of fasting.</p><p>Data expressed as Mean ± SEM. *p<0.05 vs. LDLR^−/− mice with vehicle.</p><p>Serum lipid levels in LDLR^−/− mice treated with INT-767.</p