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. 2022 Jul 26;146(21):1591–1609. doi: 10.1161/CIRCULATIONAHA.121.057623

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© 2022 The Authors.

Circulation is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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Figure 1. — NR1D1 is upregulated in human and murine aortic SMCs from AAA tissues. A, Relative mRNA expression of 49 NRs measured by Nanostring profiling in saline- or AngII-induced AAA mice (n=5 per group). The raw data were normalized to the mean expression levels of the housekeeping genes, and the normalized data were log2-transformed. The log2 (fold change) and P values were calculated, and multiple testing using the Benjamini-Hochberg method was applied to adjust the P values. With a Benjamini-Hochberg adjusted P<0.05 and |log2(fold change)|≥1 as the criteria, 3 genes (Nr1d1 [adjusted P=0.03], Nr4a1 [adjusted P=0.04], and Nr1f3 [adjusted P=0.04]) were identified as differentially expressed NRs in mouse AAA tissues. *Benjamini–Hochberg adjusted P<0.05. B, Top, Quantification of NR1D1 mRNA and NR1D1 protein expression measured by RT-qPCR and Western blot in human AAA and non-AAA segments (n=6 per group). Data were analyzed by unequal variance t test. *P<0.05; ***P<0.001. Bottom, NR1D1 protein expression assessed by Western blot in human AAA and non-AAA segments. C, Representative images of NR1D1 expression by immunofluorescence staining of human AAA and non-AAA segments and costaining with the key SMC-associated marker αSMA and DAPI. D, Top, Quantification of Nr1d1 mRNA and NR1D1 protein expression measured by RT-qPCR and Western blot in mouse abdominal aortic segments (n=6 per group). Data were analyzed by Student t test. ***P<0.001. Bottom, NR1D1 protein expression assessed by Western blot in mouse abdominal aortic segments. E, Representative images of NR1D1 expression by immunofluorescence staining in mouse suprarenal abdominal aortas and costaining with αSMA and DAPI. F, Top, Quantification of Nr1d1 mRNA and NR1D1 protein expression measured by RT-qPCR and Western blot in isolated MASMCs after stimulation with PBS or AngII for 48 hours (n=6 independent experiments). Data were analyzed by Student t test. ***P<0.001. Bottom, NR1D1 protein expression assessed by Western blot in isolated MASMCs after stimulation with PBS or AngII for 48 hours. G, Representative images of NR1D1 expression by immunofluorescence staining in MASMCs and costaining with αSMA and DAPI. Data are expressed as mean ± SEM. AAA indicates abdominal aortic aneurysm; AngII, angiotensin II; DAPI, 4’6-diamidino-2-phenylindole; αSMA, α-smooth muscle actin; MASMCs, mouse aortic smooth muscle cells; NR1D1, nuclear receptor subfamily 1 group D member 1; Nr1f3, nuclear receptor subfamily 1 group F member 3; Nr4a1, nuclear receptor subfamily 4 group A member 1 NRs, nuclear receptors; PBS‚ phosphate-buffered saline; RT-qPCR, real-time quantitative polymerase chain reaction; and SMC, smooth muscle cell.