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. 2018 Mar;38(3):555-565.
doi: 10.1161/ATVBAHA.117.310502. Epub 2018 Jan 11.

FOXO3a (Forkhead Transcription Factor O Subfamily Member 3a) Links Vascular Smooth Muscle Cell Apoptosis, Matrix Breakdown, Atherosclerosis, and Vascular Remodeling Through a Novel Pathway Involving MMP13 (Matrix Metalloproteinase 13)

Haixiang Yu  1 , Adam Fellows  1 , Kirsty Foote  1 , Zhaoqing Yang  1 , Nichola Figg  1 , Trevor Littlewood  1 , Martin Bennett  2
Affiliations

FOXO3a (Forkhead Transcription Factor O Subfamily Member 3a) Links Vascular Smooth Muscle Cell Apoptosis, Matrix Breakdown, Atherosclerosis, and Vascular Remodeling Through a Novel Pathway Involving MMP13 (Matrix Metalloproteinase 13)

Haixiang Yu et al. Arterioscler Thromb Vasc Biol. 2018 Mar.

Abstract

Objective: Vascular smooth muscle cell (VSMC) apoptosis accelerates atherosclerosis and promotes breakdown of the extracellular matrix, but the mechanistic links between these 2 processes are unknown. The forkhead protein FOXO3a (forkhead transcription factor O subfamily member 3a) is activated in human atherosclerosis and induces a range of proapoptotic and other transcriptional targets. We, therefore, determined the mechanisms and consequences of FOXO3a activation in atherosclerosis and arterial remodeling after injury.

Approach and results: Expression of a conditional FOXO3a allele (FOXO3aA3ER) potently induced VSMC apoptosis, expression and activation of MMP13 (matrix metalloproteinase 13), and downregulation of endogenous TIMPs (tissue inhibitors of MMPs). mmp13 and mmp2 were direct FOXO3a transcriptional targets in VSMCs. Activation of endogenous FOXO3a also induced MMP13, extracellular matrix degradation, and apoptosis, and MMP13-specific inhibitors and fibronectin reduced FOXO3a-mediated apoptosis. FOXO3a activation in mice with VSMC-restricted FOXO3aA3ER induced MMP13 expression and activity and medial VSMC apoptosis. FOXO3a activation in FOXO3aA3ER/ApoE-/- (apolipoprotein E deficient) mice increased atherosclerosis, increased necrotic core and reduced fibrous cap areas, and induced features of medial degeneration. After carotid artery ligation, FOXO3a activation increased VSMC apoptosis, VSMC proliferation, and neointima formation, all of which were reduced by MMP13 inhibition.

Conclusions: FOXO3a activation induces VSMC apoptosis and extracellular matrix breakdown, in part, because of transcriptional activation of MMP13. FOXO3a activation promotes atherosclerosis and medial degeneration and increases neointima after injury that is partly dependent on MMP13. FOXO3a-induced MMP activation represents a direct mechanistic link between VSMC apoptosis and matrix breakdown in vascular disease.

Keywords: apoptosis; atherosclerosis; downregulation; extracellular matrix; humans.

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Figures

Figure 1.
Figure 1.
FOXO3a (forkhead transcription factor O subfamily member 3a) activation stimulates MMP (matrix metalloproteinase) expression and activation. A, Quantitative polymerase chain reaction for mmp13 mRNA of wild-type (WT) or FOXO3aA3ER vascular smooth muscle cells (VSMCs; FOXO) treated with ethanol carrier control (−) or hydroxytamoxifen (HT; +) from 0 to 24 h. B, Western blot of conditioned media (top) and cell lysates (bottom) of FOXO3aA3ER VSMCs from 4 to 24 h. C, Zymogram of MMP activity in conditioned media from WT or FOXO3aA3ER VSMCs after carrier control (−) or HT (+) for 24 h. D, Zymogram of MMP13 activity after immunoprecipitation of conditioned medium with IgG control or MMP13 antibody after carrier control (−) or HT (+) for 24 h. E, Fluorogenic dye-quenched (DQ) gelatin fluorescence of WT or FOXO3aA3ER VSMCs treated with HT for 16 h. Insets show DAPI (4’,6-diamidino-2-phenylindole) of same field as DQ gelatin. Data are means±SD, n=3. *P<0.001 vs time 0.
Figure 2.
Figure 2.
MMP13 (matrix metalloproteinase 13) is a direct target of FOXO3a (forkhead transcription factor O subfamily member 3a). A, Dual luciferase assay of wild-type (WT) control or FOXO3aA3ER (FOXO) vascular smooth muscle cells (VSMCs) treated with carrier control (−) or hydroxytamoxifen (HT; +) for 24 h after transfection of plasmids containing a promoter construct containing a forkhead response element (FHRE) or a MMP3, 9, 13, or 2 promoter. B, Dual luciferase assay of WT or FOXO3aA3ER (FOXO) VSMCs treated with carrier control (−) or HT (+) after transfection with a −1600 MMP13 promoter fragment or after point mutation of the FOXO3a-binding site. C and D, Western blot for MMP13 in conditioned media or lysates (C), or zymography of conditioned media (D) of FOXO3aA3ER VSMCs after transfection with either control siRNA or siRNA to human FOXO3a and 24 h treatment with HT. E, Western blot of lysates from human VSMCs incubated in medium containing 10% FCS, 0% FCS (serum free [SF]), or SF and glucose-free medium (SFGF) for 24 h for phosphorylated and unphosphorylated FOXO3a. F, Western blot for MMP13 of conditioned media from mouse or human VSMCs in SF or SFGF media compared with FOXO3aER VSMCs±hydroxytamoxifen (OHT) for 24 h. G, Fluorescence microscopy for DG-gelatin of human VSMCs in E, quantified in H. I, Percentage of apoptotic cells by flow cytometry of human VSMCs in E. Data are means±SD, n=3.
Figure 3.
Figure 3.
Vascular smooth muscle cell–specific expression of FOXO3aA3ER induces apoptosis in vivo. A, Structure of the SM22αFOXO3aA3ER transgene. B, Reverse-transcriptase polymerase chain reaction for FOXO3aA3ER in aorta, heart, liver, and gut of 2 SM22αFOXO3aA3ER founder mice or a littermate control. Genomic DNA from SM22αFOXO3aA3ER or littermates were used as positive (+) or negative controls (−). C and D, Western blot (C) or immunohistochemistry (D) for FOXO3a (forkhead transcription factor O subfamily member 3a) in littermate control or SM22αFOXO3aA3ER (FOXO) mouse aortas at 7 d after tamoxifen treatment. E and F, TUNEL (terminal UTP nick-end labeling) of control or SM22αFOXO3aA3ER mouse aortas at 7 d after tamoxifen. G, Immunohistochemistry for MMP13 (matrix metalloproteinase 13) in SM22αFOXO3aA3ER mouse aortas after 5 d of vehicle control or tamoxifen. H, In situ zymography of SM22αFOXO3aA3ER mouse aortas after 5 d of tamoxifen±WAY170523 (WAY). Scale bars, 25 µm in D and 100 µm in E and G. Data are means±SD, n=6. I, Negative and positive controls for MMP13 antibody staining of lymph node. Scale bar, 50 µm. HA indicates haemagglutinin.
Figure 4.
Figure 4.
FOXO3a (forkhead transcription factor O subfamily member 3a) activation promotes atherosclerosis, increased necrotic cores, and apoptosis. Histochemistry and immunohistochemistry of aortic root plaques of control ApoE−/− or SM22αFOXO3aA3ER/ApoE−/− mice after 14 wk of fat feeding+tamoxifen. Sections were stained with H+E, Massons, or antibodies to α-SMA (smooth muscle cell α actin), mac3, or underwent TUNEL (terminal UTP nick-end labeling). Scale bar, 500 μm. Insets are high power view of area outlined in the main panel. Right, Negative control sections for SMA, mac3, and TUNEL.
Figure 5.
Figure 5.
Quantification of size and composition in mouse plaques. A and B, Plaque size and composition of aortic root (A), or brachiocephalic plaques (B) of control ApoE−/− or SM22αFOXO3aA3ER/ ApoE−/− mice after 14 wk of fat feeding. n=11 to 15 (aortic), n=8 (brachiocephalic). C–F, Plaque composition of aortic root plaques or (G) medial cell counts or (H) elastin breaks in undiseased aortas of experimental mice. Data are means±SD, n=11 to 14. FOXO indicates forkhead transcription factor O subfamily; and TUNEL, terminal UTP nick-end labeling.
Figure 6.
Figure 6.
Effects of FOXO3 (forkhead transcription factor O subfamily member 3) activation and MMP13 (matrix metalloproteinase 13) inhibition on arterial remodeling. A, Histochemistry and immunohistochemistry of left carotid artery at 28 d post-ligation of SM22αFOXO3aA3ER (FOXO) or littermate control mice, treated with tamoxifen throughout±WAY170523 or vehicle control from 0 to 7 d. Negative and positive control lymph node sections for bromodeoxyuridine (BrdU) or IgG for TUNEL (terminal UTP nick-end labeling) are shown on the (right). B, Intimal or medial areas, or percentage of cells positive for BrdU or TUNEL in both intima and media in experimental mice as in A. Data are means±SEM, n=6 to 11.
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