Age-Associated Mitochondrial Dysfunction Accelerates Atherogenesis

Abstract

Rationale: Aging is one of the strongest risk factors for atherosclerosis. Yet whether aging increases the risk of atherosclerosis independently of chronic hyperlipidemia is not known. Objective: To determine if vascular aging before the induction of hyperlipidemia enhances atherogenesis. Methods and Results: We analyzed the aortas of young and aged normolipidemic wild type, disease-free mice and found that aging led to elevated IL (interleukin)-6 levels and mitochondrial dysfunction, associated with increased mitophagy and the associated protein Parkin. In aortic tissue culture, we found evidence that with aging mitochondrial dysfunction and IL-6 exist in a positive feedback loop. We triggered acute hyperlipidemia in aged and young mice by inducing liver-specific degradation of the LDL (low-density lipoprotein) receptor combined with a 10-week western diet and found that atherogenesis was enhanced in aged wild-type mice. Hyperlipidemia further reduced mitochondrial function and increased the levels of Parkin in the aortas of aged mice but not young mice. Genetic disruption of autophagy in smooth muscle cells of young mice exposed to hyperlipidemia led to increased aortic Parkin and IL-6 levels, impaired mitochondrial function, and enhanced atherogenesis. Importantly, enhancing mitophagy in aged, hyperlipidemic mice via oral administration of spermidine prevented the increase in aortic IL-6 and Parkin, attenuated mitochondrial dysfunction, and reduced atherogenesis. Conclusions: Before hyperlipidemia, aging elevates IL-6 and impairs mitochondrial function within the aorta, associated with enhanced mitophagy and increased Parkin levels. These age-associated changes prime the vasculature to exacerbate atherogenesis upon acute hyperlipidemia. Our work implies that novel therapeutics aimed at improving vascular mitochondrial bioenergetics or reducing inflammation before hyperlipidemia may reduce age-related atherosclerosis.

Keywords: aging; atherosclerosis; cardiovascular diseases; hyperlipidemias; mitochondria.

Figure 1.. Aging leads to mitochondrial dysfunction and increased aortic Parkin expression during normolipidemia.

WT mice were maintained on a LFD until either 3-months or 18-months of age. At this point, the thoracic aorta was harvested for oxygen consumption rate (OCR) measurements, western blots or immunofluorescence staining. (A) Representative protocol for measuring the maximal OCR using inhibitors, substrates, and uncoupler (see Methods). (B) Maximal aortic OCR with substrates for complex I and I+II coupled OXPHOS and maximal complex I+II and II uncoupled OCR. (C) Aortic lysates from LFD-fed young and aged WT C57BL/6 mice were immunoblotted against PINK1, Parkin, Complex V, Complex III, and Complex II and β-actin. (D-H) Quantification of immunoblot in C. (I) Ratio of mtDNA to nuclear DNA as measured by real time PCR. (J) Fixed frozen aortic sections (6μm) were stained with primary antibodies against Parkin and smooth muscle α-actin or secondary antibodies only and nuclei were stained with hoechst. Scale bars: 10μm. (K) Mean fluorescence intensity (MFI) of parkin signal within the smooth muscle α-actin positive media layer was quantified. 2-way ANOVA with Sidak’s multiple comparison test for B. Unpaired t-test for D and E. Mann-Whitney U-test for F-I and K. Each data point represents a biological replicate. All results are presented as mean ±SEM. A=aged, CI=complex I, CII=complex II, Y=young.

Figure 2.. Aging leads to increased measures of aortic mitophagy and mitochondrial ubiquitination.

(A) Aortas from young or aged WT, LFD-fed mice were harvested and incubated with mitotracker and lysotracker then stained with hoechst and imaged by confocal microscopy. Arrows denote colocalized mitochondrial puncta and lysosomal puncta. Scale bars=10μm. (B) Fluorescence histograms of mitotracker and lysotracker signal of puncta denoted by arrows in (A) showing colocalized signal. (C) Percent of total mitochondrial puncta MFI that colocalizes with lysosome puncta (see methods). (D-E) Mitochondria were isolated from the aortas of young and aged mice and were immuoblotted against ubiquitin, β-actin, and CoxIV. Isolated mitochondria lanes were normalized against CoxIV. (F-G) Aortas were harvested from young (4 months of age) and aged (16 months of age) mtKiema mice (see methods). Aortas were incubated with mitotracker green. The mitotracker green signal (488nm excitation) and mtKeima red signal (561nm excitation) were assessed by fluorescence microscopy (see methods) and the ratio of 561:488 (mitophagy index) is shown in G. Scale bars in L= 10μm. Each data point represents a biological replicate. All results are presented as mean ±SEM. Mann-Whitney U-test for C, E, G. A=aged, Y=young.

Figure 3.. Aging leads to increased IL-6 aortic expression during normolipidemia. IL-6 impairs OCR and increases Parkin in aortic tissue culture.

(A) Thoracic aortas from young or aged WT, LFD-fed mice were harvested and frozen for immunoblots and blotted against TLR9, MyD88, IL-6, and β-actin. Quantification of immunoblots is to the right. (B) Thoracic aortas from young WT mice were incubated in DMEM+10%FBS with either 0 or 10ng/mL of IL-6 for 2h and then OCR was assessed per Figure 1A. (C) Thoracic aortas from young WT mice were incubated in DMEM+10%FBS with either 0 or 10ng/mL of IL-6 and then the lysate was immunoblotted against Parkin, Nix, and β-actin. (D) Thoracic aortas from aged (18 months of age) WT mice were incubated in DMEM+10%FBS with 5μg/ml anti-IL-6 antibody or isotype control antibody for 2h and then OCR measured. (E) Thoracic aortas from aged (18months of age) WT mice were incubated in DMEM+10%FBS with 5μg/ml anti-IL-6 antibody or isotype control for 2h and then the lysate was immunoblotted against Parkin, Nix, and β-actin. Each data point represents a biological replicate. All results are presented as mean ±SEM. Mann-Whitney U-test for A, C, and E. 2-way ANOVA with Sidak’s post-hoc test for B and D. A=aged, CI=complex I, CII=complex II, Y=young,

Figure 4.. FCCP enhances mitophagy in the aorta of aged mice in tissue culture and reduces levels of Parkin, TLR9, MyD88, and IL-6.

(A-B) Thoracic aortas from aged (16 months of age) mtKiema mice were incubated with mitotracker green and also 10μM of FCCP or vehicle control. The mitotracker green signal (488nm excitation) and mtKeima red signal (561nm excitation) were assessed by fluorescence microscopy (see methods) and the ratio of 561:488 (mitophagy index) is shown in B. (C) Thoracic aortas from 18-month old WT mice were harvested and divided into 5 equal parts and cultured in DMEM+10% FBS supplemented with the indicated concentrations of FCCP for 2h. Lysates were immunoblotted against Parkin, TLR9, MyD88, IL-6, CoxIV, and β-actin. (D-H) Quantification of immunoblots. Each point is a biological replicate. All results are presented as mean ±SEM. Kruskal-Wallis with Dunn’s post-hoc test for D-H. Mann-Whitney U-test for B.

Figure 5.. Atherosclerotic lesions show an age-associated increased size of necrosis induced by acute hyperlipidemia.

WT C57BL/6 mice were maintained on a LFD until either 3-months or 18-months and were then transfected with PCSK9-AAV and fed a WD for 10 weeks. At this point, aortic roots were obtained, paraffin-embedded, and stained with H&E. (A) Cross sections of aortic roots from male C57BL/6 mice show total lesion area, outlined in dashed lines, and necrotic core area, denoted by asterisks. Higher magnification shows the presence of necrotic core in aged mice. Scale bars: 100μm. (B and C) Quantification of total lesion area and necrotic core. (D) Cross sections of brachiocephalic artery from male C57BL/6 mice showing total lesion area, outlined in dashed lines. Scale bars: 100μm and 10μm for magnified image. (E) Quantification of total lesion area. (F) Cross sections of the aortic root were stained with Mac2 monoclonal antibody as described in detailed methods. Scale bar: 100μm. (G) Quantification of Mac2 positive staining area as a percentage of the total plaque area. Each data point represents a biological replicate. For atherosclerotic plaque and necrotic area, each biological replicate is the sum of 30 serial sections, 6μm per section. All results are presented as mean ±SEM. Student’s t-test for B, C, and G. Mann-Whitney U-test for E. A=aged, Y=young.

Figure 6.. Aged but not young mice show reduced mitochondrial function and increased levels of Parkin and PINK1 during atherogenesis.

(A) Thoracic aortas were harvested from 3-month or 18-month WT, LFD-fed mice, and WT mice maintained on a low-fat diet until either 3-months or 18-months which were then transfected with PCSK9-AAV and fed a WD for 10 weeks. Lysates were blotted against PINK1, Parkin, ATG5, Nix and β-actin (Note, that Nix immunoblot was run separately to other proteins and has its own loading β-actin control, below). (B-E) Quantification of immunoblots from A. (F) Maximal thoracic aorta OCR with substrates for complex I OXPHOS. (G): Maximal aortic OCR with substrates for complex I+II OXPHOS. (H) Maximal aortic complex I+II uncoupled OCR. (I) Maximal aortic complex II uncoupled OCR. Each data point represents a biological replicate. Note, that the normolipidemic data (i.e., WT LFD denotation) are the same data shown in Figure 1. Data from Figure 1 and Figure 6 were run contemporaneously. All results are presented as mean ±SEM. 2-way ANOVA with Sidak’s post-hoc test for B-C and F-I. Kruskal-Wallis with Dunn’s post-hoc test for E. A=aged, Y=young.

Figure 7:. Conditional loss of ATG5 in VMSCs leads to reduced mitochondrial function with increased levels of Parkin, TLR9, MyD88 and IL-6 levels during atherosclerosis.

Inducible Atg5^fl/flMyh11-cre/ER^T2 mice were treated with either tamoxifen (ATG5^−/−) or vehicle (ATG5^+/+) and some were transfected with PCSK9-AAV and fed a WD for 10 weeks before measuring proteins, mitochondrial function and aortic root sections. (A) Thoracic aortas from LFD-fed Atg5^fl/flMyh11-cre/ER^T2 mice treated with tamoxifen or littermate controls treated with vehicle were harvested and blotted against PINK1, Parkin, TLR9, MyD88, IL-6, and β-actin. (B) Quantification of immunoblots. (C) Atg5^fl/flMyh11-cre/ER^T2 mice were treated with tamoxifen or littermate controls treated with vehicle and all mice were then transfected with PCSK9-AAV and fed a WD for 10-weeks. Aortas were harvested and blotted against PINK1, Parkin, TLR9, MyD88, IL-6, and β-actin. (D) Quantification of immunoblots. (E) Maximal thoracic aorta OCR with substrates for complex I and I+II coupled OXPHOS and maximal complex I+II and II uncoupled OCR. (F) Cross sections of aortic root show total lesion area, outlined in dashed lines; and necrotic core area, outlined in dotted lines and denoted by asterisks. Scale bars: 100μm. (G and H) Quantification of total lesion area and necrotic core. Each data point represents a biological replicate. For atherosclerotic plaque and necrotic area, each biological replicate is the sum of 30 serial sections, 6μm per section. All results are presented as mean ±SEM. Mann-Whitney U-test for B, D, G, and H. 2-way ANOVA with Sidak’s multiple comparison test for E. CI=complex I, CII=complex II, Tam=tamoxifen, Veh=vehicle

Figure 8:. Spermidine treatment in aged mice mitigates increased Parkin and IL-6 levels, and mitochondrial dysfunction during atherogenesis.

WT aged mice were maintained on a LFD until 18-months old and some were then transfected with PCSK9-AAV and fed a WD for 10 weeks. During the WD feeding period, a subset of randomly selected aged mice were supplemented with spermidine in the drinking water. Some aged mice were maintained on a LFD from 18-months and treated with spermidine for 10-weeks. (A) Schematic of experimental design for B and C. (B) Thoracic aortas from aged WT LFD-fed mice either treated with spermidine or vehicle were harvested and blotted against PINK1, Parkin, TLR9, MyD88, IL-6, and β-actin. (C) Quantification of immunoblots from B. (D) Schematic of experimental design for E-J. (E) Aortas from aged WT mice treated with PCSK9-AAV and fed a WD were either treated with spermidine or vehicle and blotted against PINK1, Parkin, TLR9, MyD88, IL-6, and β-actin. (F) Quantification of immunoblots from E. (G) Maximal thoracic aortic OCR with substrates for complex I and I+II coupled OXPHOS and maximal complex I+II and II uncoupled OCR. (H) Cross sections of aortic root show total lesion area, outlined in dashed lines, and necrotic core area, outlined in dotted lines. Scale bars: 100μm. (I and J) Quantification of total lesion area and necrotic core based on H&E. Scale bars: 100μm. Each data point represents a biological replicate. For atherosclerotic plaque and necrotic area, each biological replicate is the sum of 30 serial sections, 6μm per section. All results are presented as mean ±SEM. Mann-Whitney U-test for C, F, I, and J. 2-way ANOVA with Sidak’s multiple comparison test for G. CI=complex I, CII=complex II, Sp=spermidine, Veh=vehicle.