Macrophage EP4 deficiency increases apoptosis and suppresses early atherosclerosis

Abstract

Prostaglandin (PG) E(2), a major product of activated macrophages, has been implicated in atherosclerosis and plaque rupture. The PGE(2) receptors, EP2 and EP4, are expressed in atherosclerotic lesions and are known to inhibit apoptosis in cancer cells. To examine the roles of macrophage EP4 and EP2 in apoptosis and early atherosclerosis, fetal liver cell transplantation was used to generate LDLR(-/-) mice chimeric for EP2(-/-) or EP4(-/-) hematopoietic cells. After 8 weeks on a Western diet, EP4(-/-) --> LDLR(-/-) mice, but not EP2(-/-) --> LDLR(-/-) mice, had significantly reduced aortic atherosclerosis with increased apoptotic cells in the lesions. EP4(-/-) peritoneal macrophages had increased sensitivity to proapoptotic stimuli, including palmitic acid and free cholesterol loading, which was accompanied by suppression of activity of p-Akt, p-Bad, and NF-kappaB-regulated genes. Thus, EP4 deficiency inhibits the PI3K/Akt and NF-kappaB pathways compromising macrophage survival and suppressing early atherosclerosis, identifying macrophage EP4-signaling pathways as molecular targets for modulating the development of atherosclerosis.

Figure 1. EP2 deficiency causes a compensatory increases in EP4 gene expression and EP4 deficiency suppress COX-2 production in peritoneal macrophages

(A–D) EP4(A), EP2(B), COX-1(C) and COX-2(D) gene expression levels in peritoneal macrophages isolated from LDLR^−/− mice reconstituted with WT(▪), EP4^−/−(□) or EP2^−/−( ) FLC and fed with the Western diet for 8 weeks. Macrophages were treated with media alone (control) or with LPS (50ng/ml) for 5 hours. The gene expression levels were measured by real-time PCR. Graphs represent data (Mean ± SEM) with the same number (n=3) of mice per group (*p<0.05 between control and treated with LPS cells of the same group, and between WT and EP4^−/− cells by One Way ANOVA analysis). (E,F) COX-1 (E) and COX-2 (F) protein levels in peritoneal macrophages. Macrophages were treated with media alone or with LPS for 5 hours. Cell extract (20μg/line) was resolved on 10% Bis-Tris gel and analyzed by Western blot.

Figure 2. EP4 deficiency in hematopoietic cells does not affect plasma lipid levels but significantly suppresses early atherosclerosis

(A) FPLC profiles in LDLR^−/− mice reconstituted WT(•), EP4^−/−(○), or EP2^−/−( ) FLCs. The data are represented as the average of total cholesterol of mice (n=3 per group) reconstituted different FLC and fed with the Western diet for 8 weeks. Fractions 14–17 contain VLDL; fractions 18–24 are IDL/LDL; and fractions 25–30 contain HDL. (B–D) Atherosclerotic lesions in aorta en face (B) and extent of atherosclerotic lesion area in the distal (C) and proximal (D) aortas of LDLR^−/− mice reconstituted WT(•), EP4^−/−(○), or EP2^−/−( ) FLCs. Graphs represent data (Mean ± SEM) with different number (n=12, 13 and 13, respectively) mice of each genotype (p<0.05 between control and reconstituted with EP4^−/− FLC groups by One Way ANOVA analysis, the Tukey test).

Figure 3. EP4 deficiency in hematopoietic cells increases apoptosis in atherosclerotic lesions

(A) Distribution of TUNEL+ cells in the proximal aorta of mice reconstituted with different FLC and fed with the Western diet for 8 weeks (20x magnification). The scale bars represent 20μm. (B,C) Percent of TUNEL+ cells (B) and numbers of DAPI-stained nucleus cells/MOMA-2+ area (C) in atherosclerotic lesions of LDLR^−/− mice reconstituted with WT(▪), EP4^−/−(□) or EP2^−/−( ) FLC and fed with the Western diet for 8 weeks. Graphs represent data (Mean ± SEM) with different number (n=12, 13 and 13, respectively) mice per group (*p<0.05 between mice reconstituted with WT and EP4^−/− cells by One Way ANOVA analysis).

Figure 4. EP4 deficiency in macrophages increases susceptibility to apoptosis and suppresses Akt and Bad phosphorylation

(A–D) Detection of TUNEL+ cells in untreated WT(A) and treated with PA-BSA (0.5 mM for 18 hours) WT(B), EP4^−/−(C) or EP2^−/−(D) macrophages (x20). (E, F) Percent of TUNEL+ cells in WT(▪), EP4^−/−(□) or EP2^−/−( ) macrophages untreated or treated with PA-BSA (E), oxLDL (100μg/ml) or AcLDL (100μg/ml) plus an ACAT inhibitor, Sandoz 58035 (10μg/ml) (F) for 24 hours. Graphs represent data (Mean ± SEM) with the same number (n=3) of mice per group (*p<0.05 between WT and EP4^−/− cells by One Way ANOVA analysis) (G,H) Expression of Akt, p-Akt(Ser473), and p-GSK3α/β(H), p-Bad(Ser136 and 155) and β-actin (J) in WT, EP4^−/− or EP2^−/− peritoneal macrophages untreated and treated with PA-BSA (0.5mM) for 3,6 or 18 hours. Cell extract (100μg/line) was resolved and analyzed by Western blot using antibodies against the proteins as indicated.

Figure 5. Inhibition of the PI3K/Akt signaling pathway in WT macrophages accelerates apoptosis

(A) Expression of Akt, p-Akt(Ser 473), Bad(Ser 136 and 155) and β-actin proteins in WT macrophages treated with PA-BSA (0.5mM) alone or with dibutyryl cAMP (1mM), RO-20-1724 (100μM) or, PKA inhibitor, H89 (10μM), for 3 and 6 hours. (B) Expression of Akt, p-Akt, GKS3α/β and β-actin proteins in WT macrophages treated with PA-BSA alone or with wortmannin (Wrt, 100nM) for 3 or 6 hours. Extracted proteins (100μg/lane) were resolved and analyzed by Western blot using antibodies against the proteins as indicated. (C–F) Apoptosis in WT macrophages untreated (C) or treated with Wrt(50nM; D) or PA-BSA(E) alone or in combination with Wrt(F) macrophages for 18 hours (x20). The scale bars represent 20 μm (G) Percent of apoptotic macrophages untreated and treated with Wrt (50nM; D), PA-BSA alone or in combination with Wrt for 18 hours. Graphs represent data (Mean ± SEM) with the same number (n=3) of mice per group (*p<0.05 between untreated cells and treated with PA-BSA alone and with Wrt by One Way ANOVA analysis). (H–K) Apoptosis in WT macrophages untreated(H) or treated with the Akt inhibitor IV (10μM; I) or PA-BSA(J) alone or in combination with the Akt inhibitor IV(K) for 24 hours (x20). The scale bars represent 20 μm (L) Percent of apoptotic macrophages untreated and treated with the Akt inhibitor IV (10μM), PA-BSA alone or in combination with the Akt inhibitor IV for 24 hours (*p<0.05 between cells treated with PA-BSA alone and with the Akt inhibitor by One Way ANOVA analysis, the Tukey test). (M) Expression of Akt, p-Akt, and β-actin in WT macrophages treated with Akt inhibitor IV or PABSA alone or incombination for 6 hours. Extracted proteins (100μg/lane) were resolved and analyzed by Western blot using antibodies against the proteins as indicated.

Figure 6

EP4 deficiency in macrophages suppresses NF-kB-related gene and protein expression levels. (A–I). Expression levels of inflammatory (IL1β, IL6, MCP1, MMP9), NF-κB-related (p-50, IKKα, IKKβ) and anti-apoptotic (Gadd45β and Itch) genes in WT(▪), EP4^−/−(□) or EP2^−/−( ) macrophages. The cells were treated with LPS (50nM) for 5 hours and gene expression levels were measured by real-time PCR. Graphs represent data (Mean ± SEM) of two experiments with the same number (n=3) mice per group (*p<0.05 between WT and EP4^−/− cells by One Way ANOVA analysis). (J). Protein expression levels in WT and EP4^−/− macrophages treated with PA-BSA (500 μM) for 3 and 6 hours. Cell extract (100μg/line) was resolved and analyzed by Western blot. The graph represents the data of two different experiments.

Figure 7

A schematic presentation of the EP2 and EP4 receptor signaling pathways specific for mouse peritoneal macrophages in relevance to apoptosis.