FoxO4 promotes early inflammatory response upon myocardial infarction via endothelial Arg1

Abstract

Rationale: Inflammation in post-myocardial infarction (MI) is necessary for myocyte repair and wound healing. Unfortunately, it is also a key component of subsequent heart failure pathology. Transcription factor forkhead box O4 (FoxO4) regulates a variety of biological processes, including inflammation. However, its role in MI remains unknown.

Objective: To test the hypothesis that FoxO4 promotes early post-MI inflammation via endothelial arginase 1 (Arg1).

Methods and results: We induced MI in wild-type and FoxO4(-/-) mice. FoxO4(-/-) mice had a significantly higher post-MI survival, better cardiac function, and reduced infarct size. FoxO4(-/-) hearts had significantly fewer neutrophils, reduced expression of cytokines, and competitive nitric oxide synthase inhibitor Arg1. We generated conditional FoxO4 knockout mice with FoxO4 deleted in cardiac mycoytes or endothelial cells. FoxO4 endothelial cell-specific knockout mice showed significant post-MI improvement of cardiac function and reduction of neutrophil accumulation and cytokine expression, whereas FoxO4 cardiac mycoyte-specific knockout mice had no significant difference in cardiac function and post-MI inflammation from those of control littermates. FoxO4 binds the Foxo-binding site in the Arg1 promoter and activates Arg1 transcription. FoxO4 knockdown in human aortic endothelial cells upregulated nitric oxide on ischemia and suppressed monocyte adhesion that can be reversed by ectopic-expression of Arg1. Furthermore, chemical inhibition of Arg1 in wild-type mice had similar cardioprotection and reduced inflammation after MI as FoxO4 inactivation and administration of nitric oxide synthase inhibitor to FoxO4 KO mice reversed the beneficial effects of FoxO4 deletion on post-MI cardiac function.

Conclusions: FoxO4 activates Arg1 transcription in endothelial cells in response to MI, leading to downregulation of nitric oxide and upregulation of neutrophil infiltration to the infarct area.

Keywords: FoxO4 protein, mouse; arginase; endothelial cells; inflammation; myocardial infarction.

Figure 1. FoxO4 deletion protects mice against MI-injury

WT and FoxO4 KO mice underwent permanent ligation of LAD. (A) Survival curves after MI. (B) Fractional shortening (FS) and (C) left ventricular internal diameter at systole (LVIDs) were measured by echocardiograph (n=6–12). (D) Representative images of Masson’s Trichrome-stained transverse cross sections of hearts 7 days post-MI. Sections were cut at the levels indicated. (E) Infarct size quantified from sections shown in D (n=5). (F) HW/BW ratios of mice 7 days after MI (n=5–7 for each group). *, WT vs KO, p<0.05..

Figure 2. Deletion of FoxO4 resulted in attenuated early post-MI inflammation

(A) Heat map of differentially expressed genes (>2-fold changes) in WT and FoxO4-nll mouse hearts at sham and post-MI 1 and 3 days. (B) Gene ontology analysis showing the biological processes and molecular functions of differentially expressed genes in (A). (C) qRT-PCR of inflammatory genes that are most attenuated in one-day post-MI FoxO4 KO mice. Expression was normalized against GAPDH and expressed relative to that of WT_Sham (Sh) heart (n=5). *, p<0.05 compared to WT_MI. (D) Representative images of Ly6G staining of sections of WT and FoxO4 KO mouse hearts at one day post-MI. Ly6G staining was quantified by Image-J and expressed relative to the mean value for WT mouse hearts (n=5–7). *, p< 0.05. (E) FACS profiles of non- cardiac mycoytes labeled with Ly6G (n=5–7), *, p<0.05.

Figure 3. Inactivation of FoxO4 in endothelial cells but not in cardiac mycoytes downregulates post-MI inflammation and preserves post-infarct cardiac function

(A, B) Fractional shortenings of (A) FoxO4 cKO (FoxO4^f/f;_MHC-cre) and (B) FoxO4 ecKO mice (FoxO4^f/f;Tie2-cre) and their respective control littermates (FoxO4^f/f) before and after MI (n=6–8). *, p<0.05. (C) qRT-PCR of selective cytokines from remote area (RA) and infarct area (IA) of FoxO4 ecKO and control littermates at post-MI day 1. (D) FACS profile of non- cardiac mycoytes labeled with Ly6G from hearts of FoxO4 ecKO and control littermates post-MI 2 days. Neutrophil fractions gated on the two left panel were quantified (right panel) (n=3). *, p<0.05.

Figure 4. Upregulation of post-MI Arg1 expression was significantly attenuated in FoxO4 KO and FoxO4 ecKO mouse hearts

(A) Relative mRNA of Arg1 in the remote (RA) and infarct area (IA) of FoxO4 KO and WT littermate mouse hearts post-MI 1 day as measured by qRT-PCR. mRNAs were normalized against internal GAPDH (n=6). *, p<0.05. (B) Western blot of Arg1 from tissues described in (A) (n=3). *, p<0.05. Protein levels were quantified by densitometry and normalized against GAPDH. (C) Immunofluorescent micrographs of IA from histological heart sections of FoxO4 ecKO and control littermates stained with Arg1 antibody post-MI day 2. Arg1 immunofluorescence from 5 different sections of each genotype mouse was quantified by Image-J, and averaged values are shown (n=3) *, p<0.05. Scale bar=100μM. (D) Arginase activity of lysates from remote area (RA) and infarct area (IA) of FoxO4 ecKO or control littermates (n=3). *, p<0.05.

Figure 5. FoxO4 knockdown upregulates NO and suppresses monocyte adhesion that can be rescued by ectopic expression of Arg1

(A) GFP-FoxO4 was transduced into the HAECS and stimulated with or without ischemia +TNFα. (B) HAECs cells transfected with control or FoxO4 siRNA were incubated with DAF-FM DA to visualize NO production and stimulated with ischemia for 1 hr. (C) Representative micrographs of monocyte adhesion to HAECs that were transfected with control, FoxO4, or Arg1 siRNA and stimulated with or without TNFα (D) Monocyte adhesions in (C) were quantified and averaged from 4 randomly chosen fields, and expressed as percentage relative to cells transfected with control siRNA-transfected and stimulated with TNFα (n=3). *, p<0.05. (D) Control or FoxO4 siRNA-transfected HAECs were transduced with lentiviruses expressing GFP, Arg1, or FoxO4 before adhesion assays were performed in the presence or absence of TNFα. Representative micrographs from multiple experiments (N>3) and two independent siRNA duplexes were shown. (E) Monocyte adhesion from (D) was quantified and expressed as percentage relative to that of ctl-siRNA transfected and TNFα/GFP-treated cells. (n=3). *, p<0.05.

Figure 6. FoxO4 activates Arg1 transcription

(A) Arg1 promoter (4.8kb)-driven luciferase (Luc) reporter construct (left panel) or 3xIRS-luc (right panel) were co- transfected into 293A cells with the indicated FoxO expression plasmids and internal control CMV-LacZ. The luciferase activities were normalized against co-transfected β-galactosidase and expressed relative to that from vector Flag-pcDNA transfected cells (n=3). (B) One potential Sp1- (open circle) and three FoxO4-binding sites (solid circles) were identified in the Arg1 promoter. Arg1-luc reporters with different 5’ends and point mutations in the Sp1- (m2) and FoxO4-binding site (m1) were used to identify the functional Sp1 and FoxO4 binding sites in the Arg1 promoter (n=3). (C) A gel shift assay was performed with ³²P-labeled oligonucleotide probe containing the Sp1 and FoxO4-binding sequence in the Arg1 promoter and lysates of cells transfected with Flag-FoxO proteins as indicated.

Figure 7. Arg1 may mediate the pathological function of FoxO4

(A) WT mice were given IV vehicle PBS or the arginase inhibitor BEC at one day before, the day of MI, and one day after MI and sham surgeries. The arginase inhibitor had a similar cardioprotective effect post-MI (filled-diamond with solid line vs open-square with solid line) as inactivation of FoxO4 (open-square with solid line in D vs open-square with sold line in A) (n=5–8). *, BEC vs PBS-treated groups following MI. *, p<0.05. (B) mRNA of selective cytokines expressed in RA and IA of post-MI day 1 WT mouse hearts with or without BEC treatment (n=6–7), *, p<0.05, BEC vs PBS-treated groups. (C) FACS profiles of neutrophils from post-MI day 2 WT mouse hearts treated with or without BEC (n=5–7), *, p<0.05. (D) FoxO4 KO mice were given IV PBS or the NOS inhibitor L-NAME as the same time points as (A). The improved cardiac function due to inactivation of FoxO4 over that of WT mice (open-square with solid line in D vs open-square with solid line in A) is significantly reversed by L-NAME (filled diamond with solid line in D) (n=4–5). *, p<0.05. PBS vs L-NAME treated groups after MI.