Oxidized phospholipids facilitate calcific aortic valve disease by elevating ATF4 through the PERK/eIF2α axis

Figures

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  Figure 1. Regulation of OxPL-induced macrophages on osteogenic differentiation in VICs. (A) OxPL was used to stimulate mouse macrophages RAW264.7, and RT-qPCR to detect the expression of M1 macrophage related factors. (B) Mouse macrophages RAW264.7 stimulated by OxPL were co-cultured with VICs for 7 days, and Western blot was used to detect the expression of ATF4 and osteogenic factors BMP2, OPN, and osteocalcin in VICs. (C) IF detection of osteogenic factors BMP2, OPN, and osteocalcin expression in VICs. (D) Mouse macrophages RAW264.7 stimulated by OxPL were co-cultured with VICs for 7 days to detect the activity of ALP in VICs. *p < 0.05. Cell experiments were repeated three times independently. Measurement data were expressed as mean ± standard deviation. Data obeying normal distribution and homogeneity of variance between two groups were analyzed using unpaired t-test.

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  Figure 2. Abundant expression of ATF4 in CAVD samples. (A) A heat map of differentially expressed gene expression in 2 normal samples and 2 CAVD samples in the GSE159832 dataset. (B) Venn diagram of the differentially expressed genes in the GSE159832 dataset, CAVD-related transcription factors from the JASPAR database, and the top 1500 CAVD-related genes from the GeneCards database. (C) A box plot of ATF4 expression in 2 normal samples (blue box) and 2 CAVD samples (red box) in the GSE159832 dataset. p = 0.0314. *p < 0.05.

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  Figure 3. ATF4 knockdown delays the osteogenic differentiation of VICs. (A) IF staining of α-SMA and vimentin proteins in the VICs isolated from non-calcified valve tissues. (B) RT-qPCR detection of BMP2, OPN, and osteocalcin expression in the VICs induced by OIM and normal culture medium. (C) ALP activity in the OIM-induced VICs. (D) RT-qPCR detection of ATF4 expression in the VICs induced by OIM. (E) Western blot analysis of ATF4 protein in the OIM-induced VICs. (F) RT-qPCR detection of ATF4 expression in the OIM-induced VICs treated with sh-ATF4. (G) RT-qPCR detection of BMP2, OPN, and osteocalcin expression in the OIM-induced VICs treated with sh-ATF4. (H) ALP activity in the OIM-induced VICs treated with sh-ATF4. *p < 0.05. Cell experiments were repeated three times independently. Measurement data were expressed as mean ± standard deviation. Data obeying normal distribution and homogeneity of variance between two groups were analyzed using unpaired t-test.

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  Figure 4. ATF4 silencing represses AVC in mice. (A) ARS staining of aortic valve leaflet calcification in HC-induced mice treated with sh-ATF4. (B) Expression of ATF4 in the aortic valve tissue of HC-induced mice treated with sh-ATF4 detected by RT-qPCR. (C–E) Expression of BMP2, OPN, and osteocalcin in the aortic valve tissue of HC-induced mice treated with sh-ATF4 detected by RT-qPCR, IHC, and IF. (F) IHC of CD86 protein in the aortic valve tissue of HC-induced mice treated with sh-ATF4. *p < 0.05. n = 8. Measurement data were expressed as mean ± standard deviation. Data obeying normal distribution and homogeneity of variance among multiple groups were analyzed by one-way ANOVA with Tukey's post hoc tests.

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  Figure 5. OxPAPC-polarized macrophages promote osteogenic differentiation of VICs through ATF4. OxPAPC- or DMSO- polarized macrophages were co-cultured with VICs transduced with sh-ATF4. (A) The protein expression of BMP2, OPN and osteocalcin detected by Western blot. (B) The protein expression of BMP2, OPN and osteocalcin detected by IF. (C) ALP activity determination. *p < 0.05. n = 8. Cell experiments were repeated three times independently. Measurement data were expressed as mean ± standard deviation. Data obeying normal distribution and homogeneity of variance between two groups were analyzed using unpaired t-test. Data obeying normal distribution and homogeneity of variance among multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc tests.

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  Figure 6. OxPL up-regulates ATF4 expression through PERK/eIF2α pathway. (A) OxPAPC polarized macrophages and VICs were co-cultured, followed by addition of PERK inhibitor, and the phosphorylation levels of PERK and eIF2α as well as ATF4 expression in VICs was detected by Western blot. (B) ALP activity determination. (C) BMP2, α-SMA and OPN expression in VICs was detected by IF. *p < 0.05. Cell experiments were repeated three times independently. Measurement data were expressed as mean ± standard deviation. Data obeying normal distribution and homogeneity of variance between two groups were analyzed using unpaired t-test, while those among multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc tests.

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  Figure 7. Molecular mechanism underlying the role of OxPL in CAVD. OxPL promotes the M1 polarization of macrophages by up-regulating the expression of ATF4, thereby enhancing the osteogenic differentiation of VICs and formation of AVC, ultimately promoting the occurrence of CAVD.