Dynamin-Related Protein 1 Binding Partners MiD49 and MiD51 Increased Mitochondrial Fission In Vitro and Atherosclerosis in High-Fat-Diet-Fed ApoE-/- Mice

Abstract

Novel components of the mitochondrial fission machinery, mitochondrial dynamics proteins of 49 kDa (MiD49) and 51 kDa (MiD51), have been recently described, and their potential therapeutic targets for treating cardiovascular disease have been shown, including acute myocardial infarction (AMI), anthracycline cardiomyopathy and pulmonary arterial hypertension (PAH). Here, we examined the role of MiD49 and MiD51 in atherosclerosis. MiD49/51 expression was increased in the aortic valve endothelial cells (ECs) of high-fat diet-induced atherosclerosis in ApoE^-/-mice and IL-8-induced human umbilical vein endothelial cells (HUVECs), which accelerated dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. Silencing MiD49/51 reduced atherosclerotic plaque size, increased collagen content, and decreased the IL-8-induced adhesion and proliferation of HUVECs. MiD51 upregulation resulted from decreased microRNA (miR)-107 expression and increased hypoxia-inducible factor-1a (HIF-1a) expression. Treatment with miR-107 mimics decreased atherosclerotic plaque size by reducing HIF-1α and MiD51 production. Both MiD49 and MiD51 were involved in atherosclerotic plaque formation through Drp1-mediated mitochondrial fission, and the involvement of MiD51 in this process was the result of decreased miR-107 expression and increased HIF-1α expression. The miR-107-HIF-1α-MiD51 pathway might provide new therapeutic targets for atherosclerosis.

Keywords: MiD49; MiD51; atherosclerosis; endothelial cells; mitochondrial fission.

Figure 1

Upregulation of mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51) in HUVECs and atherosclerotic ECs. (A) Representative images of Oil Red O-stained aorta from the ND (normal diet) or HFD (high-fat diet) mice that were fed for 12 weeks. (B,C) HE and Masson staining performed to show the morphology and collagen of the blood vessels; atherosclerotic plaques are delineated by dashed lines (scale bars: 200 μm). (D,E) The expression of MiD49/51 in mouse aorta detected via immunohistochemistry (IHC) (scale bars: 200 μm) and immunofluorescence (IF) (scale bars: 50 μm). (F) Representative images of mitochondria in HUVECs stimulated with or without IL-8 for 48 h. Live cells were imaged after staining with Mitotracker (Green). Mitochondrial fragmentation was analyzed using Image J 1.53c and quantified as a percentage (t-test, *** p < 0.001; n = 3; scale bars: 25 μm). (G) The protein expression levels of MiD49 and MiD51 were analyzed via Western blotting (one-way ANOVA, * p < 0.05, ** p < 0.01, **** p < 0.0001, ns: not significant; n = 3 in each group). Data are presented as medians and ranges.

Figure 1

Upregulation of mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51) in HUVECs and atherosclerotic ECs. (A) Representative images of Oil Red O-stained aorta from the ND (normal diet) or HFD (high-fat diet) mice that were fed for 12 weeks. (B,C) HE and Masson staining performed to show the morphology and collagen of the blood vessels; atherosclerotic plaques are delineated by dashed lines (scale bars: 200 μm). (D,E) The expression of MiD49/51 in mouse aorta detected via immunohistochemistry (IHC) (scale bars: 200 μm) and immunofluorescence (IF) (scale bars: 50 μm). (F) Representative images of mitochondria in HUVECs stimulated with or without IL-8 for 48 h. Live cells were imaged after staining with Mitotracker (Green). Mitochondrial fragmentation was analyzed using Image J 1.53c and quantified as a percentage (t-test, *** p < 0.001; n = 3; scale bars: 25 μm). (G) The protein expression levels of MiD49 and MiD51 were analyzed via Western blotting (one-way ANOVA, * p < 0.05, ** p < 0.01, **** p < 0.0001, ns: not significant; n = 3 in each group). Data are presented as medians and ranges.

Figure 2

MiD49 or MiD51 regulated mitochondrial network, cell proliferation, and adhesion. (A) The transfection efficiencies of the three siMiD49 or MiD51 species in the HUVECs detected via Western blotting 48 h after transfection (** p < 0.01, ns: not significant; n = 3). (B) Representative images of mitochondrial network of HUVECs stained with Mitotracker (Green). Specific small interfering RNA (siRNA) was transfected into HUVECs and induced with 50 ng/mL IL-8 for 48 h before imaging, and mitochondrial fragmentation was quantified (** p < 0.01, and *** p < 0.001; scale bars: 25 μm, n = 3). (C,F–H) HUVECs transfected with siMiD49 or siMiD51 and induced with IL-8 at 50 ng/mL. The cells were harvested 48 h later for Western blotting. Representative images of the Western blot and the densitometries of the expressions of p-Drp1_Ser616, Drp1, p-ERK1/2, ERK1/2, PCNA, Cyclin D1, p27^Kip1, VCAM-1, and ICAM-1 are shown. GAPDH or α-tubulin was used as the loading control (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; n = 3). Ctrl indicates control; siMiD is the siRNA against MiD49 or MiD51. (D) Cell proliferation analyzed 48 h after transfection by CCK8 (*** p < 0.001; n = 3). (E) Cell adhesion analyzed after the incubation of HUVECs with BCECF AM-labeled THP-1 cells for 4 h (*** p < 0.001 and **** p < 0.0001; scale bars: 200 μm; n = 3). All data are presented as medians and ranges. Statistically significant differences between groups were determined using one-way ANOVA.

Figure 2

MiD49 or MiD51 regulated mitochondrial network, cell proliferation, and adhesion. (A) The transfection efficiencies of the three siMiD49 or MiD51 species in the HUVECs detected via Western blotting 48 h after transfection (** p < 0.01, ns: not significant; n = 3). (B) Representative images of mitochondrial network of HUVECs stained with Mitotracker (Green). Specific small interfering RNA (siRNA) was transfected into HUVECs and induced with 50 ng/mL IL-8 for 48 h before imaging, and mitochondrial fragmentation was quantified (** p < 0.01, and *** p < 0.001; scale bars: 25 μm, n = 3). (C,F–H) HUVECs transfected with siMiD49 or siMiD51 and induced with IL-8 at 50 ng/mL. The cells were harvested 48 h later for Western blotting. Representative images of the Western blot and the densitometries of the expressions of p-Drp1_Ser616, Drp1, p-ERK1/2, ERK1/2, PCNA, Cyclin D1, p27^Kip1, VCAM-1, and ICAM-1 are shown. GAPDH or α-tubulin was used as the loading control (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; n = 3). Ctrl indicates control; siMiD is the siRNA against MiD49 or MiD51. (D) Cell proliferation analyzed 48 h after transfection by CCK8 (*** p < 0.001; n = 3). (E) Cell adhesion analyzed after the incubation of HUVECs with BCECF AM-labeled THP-1 cells for 4 h (*** p < 0.001 and **** p < 0.0001; scale bars: 200 μm; n = 3). All data are presented as medians and ranges. Statistically significant differences between groups were determined using one-way ANOVA.

Figure 3

Downregulation of miR-107 mediates MiD51 upregulation by targeting HIF-1α in IL-8-induced HUVECs. (A,B) Quantification of miR-107 performed via quantitative reverse transcription–polymerase chain reaction (qRT-PCR) (t-test, ** p < 0.01, **** p < 0.0001; n = 3). (C) Representative images of Western blotting and densitometries showing the expressions of MiD49 and MiD51 in HUVECs transfected with the miR-107 mimic. Cells were transfected with miR-107 for 48 h. GAPDH was used as the loading control (t-test, *** p < 0.001, ns: not significant; n = 3). (D) Representative images of mitochondrial networks of HUVECs transfected with miR-107 mimic and induced with 50 ng/mL of IL-8 for 48 h (one-way ANOVA, ** p < 0.01 and *** p < 0.001; n = 3; scale bars: 25 μm). (E) Cell proliferation analyzed 48 h after transfection using CCK8 (one-way ANOVA; *** p < 0.001 and **** p < 0.0001; n = 3). (F) Cell adhesion analyzed after the incubation of HUVECs with BCECF AM-labeled THP-1 cells (one-way ANOVA, *** p < 0.001; scale bars: 200 μm; n = 3). (G) The dual luciferase-reporter was used to determine whether or not miR-107 binds to the 3′-UTR of the MiD51 gene (WT: wild type; MUT: mutant type; two-way ANOVA, ns: not significant; n = 3). (H,I) Representative images of Western blotting and densitometries showing the expressions of HIF-1α in HUVECs transfected with the miR-107 mimic and siHIF-1α for 48 h. GAPDH was used as the loading control (one-way ANOVA, * p < 0.05 and ** p < 0.01; n = 3). (J) Representative images of Western blotting and densitometries showing the expressions of MiD51 in HUVECs transfected with siHIF-1α for 48 h. GAPDH was used as the loading control (t-test, * p < 0.05; n = 3). (K,L) Cell proliferation and adhesion analyzed 48 h after transfection with siHIF-1α (t-test, ** p < 0.01 and *** p < 0.001; n = 3, scale bars: 100 μm). (M,N) Representative images of the Western blot and the densitometries of the expressions of PCNA, Cyclin D1, p27^Kip1, VCAM-1 and ICAM-1 in HUVECs transfected with the miR-107 mimic. α-tubulin was used as the loading control (one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001; n = 3). All data are presented as medians and ranges.

Figure 3

Downregulation of miR-107 mediates MiD51 upregulation by targeting HIF-1α in IL-8-induced HUVECs. (A,B) Quantification of miR-107 performed via quantitative reverse transcription–polymerase chain reaction (qRT-PCR) (t-test, ** p < 0.01, **** p < 0.0001; n = 3). (C) Representative images of Western blotting and densitometries showing the expressions of MiD49 and MiD51 in HUVECs transfected with the miR-107 mimic. Cells were transfected with miR-107 for 48 h. GAPDH was used as the loading control (t-test, *** p < 0.001, ns: not significant; n = 3). (D) Representative images of mitochondrial networks of HUVECs transfected with miR-107 mimic and induced with 50 ng/mL of IL-8 for 48 h (one-way ANOVA, ** p < 0.01 and *** p < 0.001; n = 3; scale bars: 25 μm). (E) Cell proliferation analyzed 48 h after transfection using CCK8 (one-way ANOVA; *** p < 0.001 and **** p < 0.0001; n = 3). (F) Cell adhesion analyzed after the incubation of HUVECs with BCECF AM-labeled THP-1 cells (one-way ANOVA, *** p < 0.001; scale bars: 200 μm; n = 3). (G) The dual luciferase-reporter was used to determine whether or not miR-107 binds to the 3′-UTR of the MiD51 gene (WT: wild type; MUT: mutant type; two-way ANOVA, ns: not significant; n = 3). (H,I) Representative images of Western blotting and densitometries showing the expressions of HIF-1α in HUVECs transfected with the miR-107 mimic and siHIF-1α for 48 h. GAPDH was used as the loading control (one-way ANOVA, * p < 0.05 and ** p < 0.01; n = 3). (J) Representative images of Western blotting and densitometries showing the expressions of MiD51 in HUVECs transfected with siHIF-1α for 48 h. GAPDH was used as the loading control (t-test, * p < 0.05; n = 3). (K,L) Cell proliferation and adhesion analyzed 48 h after transfection with siHIF-1α (t-test, ** p < 0.01 and *** p < 0.001; n = 3, scale bars: 100 μm). (M,N) Representative images of the Western blot and the densitometries of the expressions of PCNA, Cyclin D1, p27^Kip1, VCAM-1 and ICAM-1 in HUVECs transfected with the miR-107 mimic. α-tubulin was used as the loading control (one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001; n = 3). All data are presented as medians and ranges.

Figure 4

Therapeutic implications of MiD49 or the miR-107–MiD51–HIF-1a pathway in the formation of atherosclerotic plaque in mice. (A) Oil Red O staining performed to show the lipid deposits of the blood vessels (n = 3). Representative images derived from normal-fed C57 mice and high-fat-diet-fed ApoE^-/- mice that were administered sh-MiD49, sh-MiD51 or agomir miR-107 (miR-107 mimic) are shown. (B) Representative images of HE stained sections. Atherosclerotic plaques are delineated by dashed lines. Scale bars: 200 µm. (C) Statistical analysis data of Oil Red O staining (up) and HE staining (down) (one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3). (D,E) The expression of MiD51 and HIF-1α in mouse aortic ECs (CD31) detected using IF (blue, DAPI; red, MiD51 or HIF-1α; green, CD31; n = 3, scale bars: 50 μm). (F) Changes in body weight of mice during treatment (t-test or one-way ANOVA; n ≥ 4). (G) Graph showing that mice were sacrificed and blood samples were taken to detect lipid levels (one-way ANOVA; ** p < 0.01, *** p < 0.001 and **** p < 0.001, ns: not significant; n ≥ 4). All data are presented as medians and ranges.

Figure 4

Therapeutic implications of MiD49 or the miR-107–MiD51–HIF-1a pathway in the formation of atherosclerotic plaque in mice. (A) Oil Red O staining performed to show the lipid deposits of the blood vessels (n = 3). Representative images derived from normal-fed C57 mice and high-fat-diet-fed ApoE^-/- mice that were administered sh-MiD49, sh-MiD51 or agomir miR-107 (miR-107 mimic) are shown. (B) Representative images of HE stained sections. Atherosclerotic plaques are delineated by dashed lines. Scale bars: 200 µm. (C) Statistical analysis data of Oil Red O staining (up) and HE staining (down) (one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3). (D,E) The expression of MiD51 and HIF-1α in mouse aortic ECs (CD31) detected using IF (blue, DAPI; red, MiD51 or HIF-1α; green, CD31; n = 3, scale bars: 50 μm). (F) Changes in body weight of mice during treatment (t-test or one-way ANOVA; n ≥ 4). (G) Graph showing that mice were sacrificed and blood samples were taken to detect lipid levels (one-way ANOVA; ** p < 0.01, *** p < 0.001 and **** p < 0.001, ns: not significant; n ≥ 4). All data are presented as medians and ranges.

Figure 5

Schematic representation of the relevance between atherosclerosis and the mitochondrial dynamic protein (MiD) pathway.