Atherosclerosis 240 (2015) 335e344
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Retinol binding protein 4 induces mitochondrial dysfunction and vascular oxidative damage Jingjing Wang a, b, 1, Hongen Chen a, b, 1, Yan Liu a, b, Wenjing Zhou b, Ruifang Sun b, Min Xia a, b, * a b
Guangdong Provincial Key Laboratory of Food, Nutrition and Health, China Department of Nutrition, School of Public Health, Sun Yat-sen University (Northern Campus), Guangzhou, Guangdong Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 November 2014 Received in revised form 8 March 2015 Accepted 25 March 2015 Available online 28 March 2015
Objectives: Mitochondrial dysfunction has been implicated in cardiovascular diseases. Elevation of serum retinol binding protein 4 (RBP4) in patients has been linked to cardiovascular disease. However, the role of RBP4 on mitochondrial oxidative stress and vascular oxidative damage is not well demonstrated. Therefore, we evaluated the impact of RBP4 on the mitochondrial reactive oxygen species (ROS) and dynamics in the pathogenesis of cardiovascular diseases. Methods and results: RBP4 treatment increased mitochondrial superoxide generation in a dosedependent manner in human aortic endothelial cells (HAECs). Exposure to RBP4 also promoted mitochondrial dysfunction as determined by decreased mitochondrial content and integrity as well as membrane potential in HAECs. Incubation with RBP4 suppressed mitofusin (Mfn)-1 protein expression, but enhanced dynamin-related protein-1 (Drp1) and fission-1 (Fis1) protein expression in HAECs, suggesting an impairment of mitochondrial fusion and fission dynamics. Moreover, RBP4 treatment significantly induced endothelial apoptosis, increased the expression of Cytochrome C and Bax, but decreased the expression of Bcl-2. Furthermore, RBP4 stimulation suppressed phosphatidyl inositol 3kinase (PI3K)/Akt signaling in HAECs. Finally, RBP4-Tg mice exhibited severe mitochondrial dysfunction and vascular oxidative damage in aorta compared with wide-type C57BL/6J mice. Conclusion: The present study uncovers a novel mechanism through which RBP4 induces vascular oxidative damage and accelerates the development of atherosclerosis. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Retinol binding protein 4 Mitochondrial dysfunction ROS Endothelial cells
1. Introduction Mitochondrial dysfunction including increased production of reactive oxygen species (ROS) in mitochondria, accumulation of mitochondrial DNA damage, and progressive respiratory chain dysfunction contributes to the pathogenesis of atherosclerosis in human investigations and animal models of oxidative stress [1,2]. There is growing appreciation of the importance of altered mitochondrial dynamics in cardiovascular disease [3,4]. Mitochondria undergo cycles of fusion to form networks and fission to form smaller individual mitochondria [5,6]. Proteins controlling fusion include mitofusin (Mfn)-1, Mfn2, and optic atropy-1 (Opa1). Fission
* Corresponding author. Department of Nutrition, School of Public Health, Sun Yat-sen University (Northern Campus), Guangzhou, Guangdong Province, China. E-mail address: [email protected] (M. Xia). 1 Jingjing Wang and Hongen Chen contribute equally to this paper. http://dx.doi.org/10.1016/j.atherosclerosis.2015.03.036 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
is regulated by dynamin-related protein-1 (Drp1) and fission-1 (Fis1). Fusion may be beneficial by allowing the distribution of metabolites, proteins, and DNA throughout the network [7]. At the end of their life cycle, dysfunctional mitochondria and damaged mitochondrial components are eliminated by fission and subsequent autophagy [8]. Under pathological conditions, including atherosclerosis, fission is increased and autophagy is impaired, leading to a loss of mitochondrial networks, accumulation of small dysfunctional mitochondria, and increase of mitochondrial ROS [9]. Retinol-binding protein 4 (RBP4), an adipokine primarily secreted by adipocytes and hepatocytes, is the sole carrier of retinol in the blood, responsible for the transportation of retinol from liver stores to peripheral tissues [10,11]. The potential link between RBP4 and insulin resistance and diabetes had been suggested by several reports showing that the RBP4 levels were elevated in insulinresistant mice and in humans with obesity and type 2 diabetes mellitus [12e14]. Recent clinical studies have linked elevated
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2. Materials and methods
peroxidation was determined by the measurement of thiobarbituric acid reactive substances (TBARS) in arteries. The freshly obtained arteries were grinded with liquid nitrogen, lysed and then 500 ml of homogenate was mixed with thiobarbituric acid and heated in boiling water for 40 min. When cooling down, the absorbance was measured at 532 nm (TECAN, Sunrise). The assay of total superoxide dismutase (SOD) activity was based on the capacity of SOD to inhibit the oxidation of WST-1 2-(4iodophenyl)-3-(4-nitrophenyl) -5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt. Activity of Cu,Zn-SOD isoform was measured by adding the inactivator of Mn-SOD and then Mn-SOD activity was calculated by subtracting Cu, Zn-SOD activity from the total SOD activity. The absorbance was measured at 550 nm (TECAN, Sunrise).
2.1. Materials
2.6. Mitochondrial membrane potential (DJm)
Recombinant human RBP4 (NP_006735.2, Met 1-Leu 201) expressed in HEK293 cells with a C-terminal polyhistidine tag was obtained from Sino Biological Inc. (Beijing, China). Endothelial Cell Medium (ECM), fetal bovine serum, endothelial cell growth supplement (ECGS) and antibiotic mixtures (Penicillin-Streptomycin) were provided by American ScienCell Technologies Inc (Carlsbad, USA).
After incubation with RBP4 for 24 h, HAECs were washed with PBS, incubated with JC-1 dye (5,5,6,6-tetrachloro-1,1,3,3tetraethylbenzimidazolyl-carbocyanine iodide) (5 mg/mL, Enzo, Farmingdale, New York, USA) for 10 min at 37 C and then observed with a confocal microscope (Leica TCS SP5 II). According to the manufacturer's instructions, two fluorescences could be captured in live cells. An orange J-aggregate emission profile was detected in healthy cells, and as membrane potential decreases, JC-1 monomers were generated, resulting in a shift to green emission. For the mitochondrial membrane potential of C57BL/6J and RBP4-Tg mice, arteries from the two groups were measured using an assay kit (GMS10013.4, GENMED, Shanghai, China). Freshly obtained arteries were washed twice with GENMED cleaner, incubated with GENMED staining at 37 C for 60 min and then lysed with GENMED lysate before images were captured by confocal microscope at 490/530 nm. According to the instructions, only green flourescence could be detected in tissues.
serum RBP4 specifically to cardiovascular disease, including hypertension [15], stroke [16], and atherosclerosis [17]. Notably, the association between higher circulating RBP4 levels and an increased risk of coronary heart disease (CHD) was confirmed among women in the Nurses' Health Study [18], indicating that RBP4 may contribute to the development of atherosclerosis. The finding that elevation of RBP4 is correlated with cardiovascular disease warrants our investigation into the possible role of RBP4 in the pathogenesis of vascular disease. The present study was designed to investigate the effect of RBP4 on mitochondrial dynamics and vascular oxidative damage in vitro and in vivo.
2.2. Experimental animals Transgenic mice expressing human RBP4 (RBP4-Tg, Stock Number: 031125- UUCD) were provided by the Mutant Mouse Regional Resource Centers (MMRRC, Rockville, North America). C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The animals (n ¼ 10 each group) were fed with a regular chow diet (Medicience Ltd, Yangzhou, China) for two months before applying for experiments (at 16 weeks of age). The animals were housed in a specific pathogenefree facility in a temperature-controlled room (22e24 C) with a 12 h lightedark cycle and given free access to food and water. The approval for this animal research was granted by the ethic review board of Sun YatSen University. 2.3. Cell culture Human aortic endothelial cells (HAECs) were obtained from ScienCell Research Laboratories and cultured with the ECM supplemented with FBS (10% v/v), penicillin (100 U/mL), streptomycin (100 mg/mL), heparin (90 mg/mL), and endothelial cell growth supplement (20 mg/mL) at 37 C under 5% CO2 atmosphere. Only three to ten passages of HAECs were used in these experiments. 2.4. Measurement of mitochondrial ROS production Cells were precultured with RBP4 for 24 h, washed with PBS, and then, stained with MitoSOX™ Red (5 mmol/L, invitrogen, Oregon, USA), a mitochondrial superoxide indicator for 10 min at 37 C, protected from light. Cells were washed gently three times with warm PBS, and then, stained with Hoechst 33258 (2 mg/mL, H21491, Invitrogen, Oregon, USA) for another 20 min at 37 C. The fluorescence of MitoSOX™ Red was detected under a confocal microscope (Leica TCS SP5Ⅱ) [19]. 2.5. Detection of SOD and MDA Superoxide dismutase (SOD) activity and MDA levels were measured using assay kits (Jiancheng Biotechnology Co. Nanjing, China) according to the manufacturers' instructions. The lipid
2.7. Mitochondrial quantification and fragmentation Cells were treated with RBP4 for 24 h, washed with PBS, then incubated with MitoTracker® Green FM (50 nM; invitrogen, Oregon, USA) for 20 min at 37 C. Cells were washed gently three times with warm PBS, and then, stained with Hoechst 33258 (2 mg/mL; H21491, Invitrogen, Oregon, USA) for another 20 min at 37 C. The fluorescence of MitoTracker® Green FM was detected under a confocal microscope (Leica TCS SP5Ⅱ) and the fluorescence intensity was quantified by Image J software. 2.8. Transmission electron microscopy A stretch of thoracic aorta was immediately removed, fixed in a solution of 2.5% glutaraldehyde, postfixed in 1% osmium teroxide for 1~2 h, and then dehydrated in gradient ethanol. The ultrathin slices were viewed with a Tecnai G2 Spirit Twin electron microscope (American FEI, Czech). 2.9. Flow cytometry Quantification of mitochondrial ROS and content in HAECs were performed using Mito-SOX and MitoTracker® Green respectively. Aliquots of 106~108 cells were centrifuged to obtain a cell pellet. Resuspending the cells gently in pre-warmed (37 C) staining solution with the fluorescent probes as described above in the dark at 37 C. After staining, the cells were re-pelleted by centrifugation and resuspended in fresh PBS, and then, a flow cytometric analysis was conducted. All analyses were performed by FACScan (BD, FACS Calibur) and the FlowJo V7.6.5 software.
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2.10. Detection of apoptosis To identify chromatin condensation, HAECs were cultured with Hoechst 33258 (2 mg/mL, H21491, invitrogen, Oregon, USA) for 30 min at 37 C. The blue fluorescent was efficiently excited with a UV laser and the stained cells were visualized using a confocal microscope at a magnification of 400. For quantification, a blind analysis from three to four randomized fields was performed. Cells were counted as apoptotic when the stained nucleus produced bright blue fluorescence due to chromatin condensation [20]. 2.11. Western blot Total protein samples were extracted from the aorta or HAECs using the RIPA lysis buffer. Aliquots (30 mg) were separated by SDSPAGE and then transferred to a PVDF membrane. After blocking with 5% BSA, the blots were incubated with various primary antibodies followed by horseradish peroxidase-labeled secondary antibodies. The protein bands were detected with a SuperSignal West Pico Chemiluminescent kit (Thermo, Massachusetts, USA). Primary antibodies for Mfn1, Mfn2, Opa-1, Drp-1 Fis-1, and GAPDH were from Santa Cruz (Texas, USA) and PI3K, phospho-PI3K, AKT, phospho-AKT (Ser473), phospho-AKT (Thr308), Cytochrome C, Bcl2 and Bax were from Cell Signaling (Boston, USA). 2.12. Statistical analysis Statistical analyses were performed with the SPSS 13.0. Data were expressed as mean ± S.E.M. from five or more independent experiments run in triplicate. All variables were tested by Shapiro-
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Wilk test for normal distribution. Student's unpaired t test was used for comparison between two groups. When more than two groups were compared One-way analysis of variance (ANOVA) was used. P < 0.05 was considered as statistically significant. 3. Results 3.1. Effect of RBP4 on the mitochondrial superoxide generation We applied the fluorescent probe MitoSOX™ Red, a superoxidesensitive fluorescent dye, to determine the effect of RBP4 on mitochondrial accumulation of superoxide by using confocal microscopy. Compared with untreated control, RBP4 treatment caused a dose-dependent increase of bright red fluorescence, indicating an enhanced mitochondrial superoxide production (Fig. 1A and B). This result was further confirmed by flow cytometry analysis, showing that RBP4 (40 mg/mL) exposure resulted in about 2.6-fold increase of fluorescence intensity compared with untreated control (Fig. 1C and D). 3.2. Effect of RBP4 on mitochondrial dysfunction in endothelial cells To investigate the effect of RBP4 on mitochondrial dysfunction, we used MitoTracker Green, a specific marker for mitochondria, to ascertain mitochondrial quantity and integrity. RBP4 exposure resulted in a concentration-dependent reduction of the green fluorescence (Fig. 2A and B), suggesting a significant decrease of mitochondrial content. This finding was further confirmed by the flow cytometry analysis, demonstrating about ~38% decrease of mitochondrial content in response to RBP4 treatment (Fig. 2C and
Fig. 1. RBP4 induced mitochondrial ROS formation. HAECs were cultured to 80% and treated with different doses of RBP4. Mitochondrial ROS production was measured by MitoSOX510/580nm and fluorescence was measured by (A) confocal microscope (magnification of 400) and (C) flow cytometry. (B) Quantitative analysis of fluorescence intensity. (D) Mean integrated fluorescence (MIFL) for Mito-SOX for each condition in (C). Results were represented as mean ± SEM of five individual experiments;*p < 0.05 vs. control (0 mg/mL), ** p < 0.01 vs. control (0 mg/mL).
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D). The proportion of fragmented versus tubular organelles was augmented in response to RBP4 treatment in a dose-dependent fashion (Fig. 2E). Mitochondrial membrane potential (DJm) is essential for normal mitochondrial function. Loss of DJm caused mitochondrial membrane depolarization and triggered a cascade of mitochondrion-dependent apoptotic signaling [21,22]. We therefore determined the effect of RBP4 on this mitochondrial parameter using JC-1 staining. Treatment of HAECs with RBP4 depolarized DJ (red fluorescence/green fluorescence) in a dose-dependent manner after 24 h incubation (Fig. 2F and G). 3.3. Effect of RBP4 on mitochondrial dynamic in endothelial cells Mitochondrial fusion and fission balance controlled the number, length and tubular shape of mitochondria [23]. Once the balance was broken, mitochondrial networks were damaged leading to mitochondrial dysfunction [24]. We thus determined the effect of RBP4 on mitochondrial fusion (Mfn1, Mfn2, Opa1) and fission (Drp1 and Fis1) proteins, which are key regulators of mitochondrial morphology. RBP4 treatment decreased the Mfn1 protein expression in a dose-dependent manner in HAECs. On the contrary, RBP4 stimulation increased the protein expression of both Drp1 and Fis1 (Fig. 2H and I). 3.4. Effect of RBP4-mediated mitochondrial dysfunction on endothelial apoptosis Mitochondrial dysfunction plays a crucial role in the regulation of the endogenous pathways of apoptosis activated by oxidative stress [25]. To determine whether RBP4- induced mitochondrial dysfunction was associated with apoptosis, we used Hoechst 33258 staining to analyze chromatin condensation, a key hallmark of apoptosis. Compared with control, RBP4 (20 mg/mL) treatment caused approximate 1.3-fold increase in apoptotic rate, as presented with intense fluorescence staining. Exposure to RBP4 treatment (40 and 60 mg/mL) induced a 2~2.5-fold increase in apoptosis cells compared to the control (Fig. 3A and B, P < 0.01). Furthermore, the apoptosis-related proteins were assessed. RBP4 treatment significantly increased the expression of Cytochrome C and Bax, but largely decreased the expression of Bcl-2 (Fig. 3C and D). 3.5. Effect of RBP4 on PI3K/AKT signaling pathway in endothelial cells It has been demonstrated that the activation of PI3K/AKT signaling pathway inhibits cellular apoptosis and promotes cell survival [26]. To determine whether RBP4 had an effect on insulin signaling in HAECs, the phosphorylation of AKT, the downstream effector of PI3K, was examined in RBP4-treated HAECs as described above. Dose-response studies demonstrated that compared to lowdose RBP4 treatment, PI3K phosphorylation was significantly decreased after treatment with high-dose RBP4. The phosphorylation of AKT at Ser473 sites was significantly decreased, while, the phosphorylation of AKT at Thr308 site had no obvious change. Total AKT and PI3K expression was not changed by any dose of RBP4 used
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in these studies (Fig. 3EeH). 3.6. Overexpression of RBP4 induces mitochondrial dysfunction and vascular oxidative damage in vivo To gain insight into the role of RBP4 in mitochondrial dysfunction and oxidative damage in vivo, we next determined its metabolic effects in the arteries of RBP4-Tg mice. The MDA production of RBP4-Tg mice arteries were ~1.6-fold higher than that of C57BL/6J (Fig. 4A, P < 0.05). Superoxide dismutases (SOD) are the major antioxidant enzymes for superoxide removal, and in mammals, three SOD isoforms are expressed: copper/zinc SOD (CuZn-SOD, SOD1), located in the cytoplasm; manganese SOD (Mn-SOD, SOD2), localized in the mitochondrial matrix; and extracellular SOD (ECSOD, SOD3). The total SOD activity from arteries of RBP4-Tg mice decreased ~20% compared with C57BL/6J (Fig. 4B, P < 0.05). MnSOD activity from mitochondria of RBP4-Tg arteries decreased ~42% compared with C57BL/6J (Fig. 4C, P < 0.05), however, CuZnSOD activity did not reduced significantly (Fig. 4D), implying that reduced antioxidant capacity was mainly attributed to the dysfunction of mitochondria. Analysis of the morphology of mitochondria by transmission electron microscope revealed a vague or absent mitochondrial crest and mitochondrial division section in RBP4-Tg mice artery (Fig. 4E). In addition, JC-1 monomers (green) were significantly highly generated in RBP4-Tg mice arteries compared with C57BL/6J (Fig. 4F and G), indicating an aggravated mitochondrial membrane depolarization in RBP4-Tg mice. RBP4-Tg mice had a lower level of mitochondrial fusion protein expression of Mfn1 than that in C57BL/6J mice (P < 0.05). Conversely, a higher level of mitochondrial fission proteins, including Drp1 and Fis-1 was observed in RBP4-Tg mice compared with C57BL/6J mice (Fig. 5A and B, P < 0.05). Furthermore, arteries from RBP4-Tg mice had a lower level of Bcl-2 and a higher level of Cytochrome C and Bax compared with C57BL/6J (Fig. 5C and D). In consistence with decreased PI3K/Akt activity in HAECs, phosphorylation of PI3K and Akt at Ser473 were significantly attenuated in arteries from RBP4-Tg mice, while phosphorylation of AKT at Thr308 site had no obvious change (Fig. 5EeH). 4. Discussion In the present study, we presented a novel mechanism by which RBP4 deteriorates endothelial mitochondrial function and promotes vascular oxidative stress. Both exogenous RBP4 treatment on HAECs and overexpression of RBP4 in mice induced mitochondrial superoxide production, impaired mitochondrial content and integrity as well as membrane potential, leading to endothelial mitochondrial dysfunction. The loss of function was due to the imbalance of fusion and fission cycle in mitochondria, with reduced Mfn1 and significantly increased Drp1 and Fis-1 expressions. RBP4induced mitochondrial dysfunction was also accompanied by apoptosis in both HAECs and RBP4-Tg mice, which was associated with impaired PI3K/AKT signaling pathway. A substantial number of studies have revealed that among the potential causes of cardiovascular diseases, oxidative stress mediated changes within the cardiovascular environment are essential
Fig. 2. RBP4 promoted mitochondrial dysfunction. Mitochondrial content was measured by MitoTracker Green and fluorescence was measured by (A) confocal microscope (magnification of 400) and (C) flow cytometry. (B) Quantitative analysis of fluorescence intensity. (D)Mean integrated fluorescence (MIFL) for MitoTracker Green for each condition in (C). (E) A shift of mitochondrial morphology from tubular to fragmented forms was observed in response to high-dose RBP4 stimulation (magnification of 1000 top and 10,000 down). (F) Reduced mitochondrial membrane potential was measured with JC-1. (G) Quantitative analysis of the ratio of JC-1 oligomer (red)/monomer (green) fluorescence. (H) Representative Western blot analysis of mitochondrial fusion and fission proteins. (I) Quantitative analysis of the protein levels. Results were represented as mean ± SEM of five individual experiments; *p < 0.05 vs. control (0 mg/mL), **p < 0.01 vs. control (0 mg/mL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Effect of RBP4-mediated mitochondrial dysfunction on endothelial apoptosis. Cultured HAECs were incubated with RBP4 for 24 h. (A) Apoptotic HAECs were calculated by Hoechst 33258346/460nm staining. Original magnification 400. (B) Apoptotic cells were counted based on morphology, and the number of apoptotic cells was expressed as percentage of total cells. (C, D) Representative Western blot and quantitative analysis of Cytochrome C, Bax, Bcl-2 protein expressions. (E) Representative Western blot analysis of PI3K, p-PI3K, AKT, p-AKTser473, p-AKTThr308 protein expressions in HAECs. (FeH) Quantitative analysis of PI3K and AKT phosphorylation. Results were represented as mean ± SEM of five individual experiments; *p < 0.05 vs. control (0 mg/mL), **p < 0.01 vs. control (0 mg/mL).
in the progression of cardiovascular diseases [27e29]. Although ROS is capable of targeting a variety of subcellular components, the mitochondrial membranes, proteins and mtDNA appear particularly sensitive to oxidative stress [30]. Increasing evidence indicates
that mitochondrial oxidative stress and the subsequently induced mitochondrial dysfunction contribute to the multiple pathological processes underlying the cardiovascular diseases. Mitochondrial integrity and functions are essential in determining individual
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Fig. 4. Overexpression of RBP4 induced vascular oxidative damage in mice. Freshly obtained arteries from C57BL/6J and RBP4-Tg mice were measured to determine (A) the TBARs levels and (BeC) the total SOD activity, Mn-SOD isoform from mitochondria and CuZn-SOD activity. (E) The ultrastructure of mitochondria in arteries of C57BL/6J and RBP4-Tg mice were observed using a transmission electron microscope (Original magnification 46,000). (F) Freshly obtained arteries of C57BL/6J and RBP4-Tg mice were stained with GENMED staining and observed at 490 nm excitation and 530 nm emission. (G) Quantitative analysis of green fluorescence intensity. Values were presented as means ± SEM, n ¼ 10 each group; *p < 0.05 vs. control (C57BL/6J), **p < 0.01 vs. control (C57BL/6J).
vulnerability to common CVD risk factors. Besides, mitochondrial damage and dysfunction have been observed in both human cells and animal models [31e33]. The link between elevation of serum RBP4 and cardiovascular diseases had been reported by several clinical studies [34e37], however the exact role of RBP4 in the pathogenesis of cardiovascular disease remains unknown. Consistent with the report that incubation with holo-RBP4 dose-dependently increases the expression of Nox2 and Nox4, leading to activation of NADPH oxidases and increase in mitochondrial O2$ [38], in the present study, we found that at cellular levels RBP4
treatment dose-dependently induced mitochondrial ROS, impaired mitochondrial content and integrity and decreased the membrane potential. A similar observation was evidenced in RBP4-Tg mice, with a higher expression of lipid peroxidation, abnormal mitochondria ultrastructure, and reduced mitochondrial antioxidant capacity in the artery. As a consequence of mitochondrial dysfunction, the negative alterations including impaired ATP production and increased ROS generation could affect the physiology and promote apoptosis in all cell types involved in cardiovascular disease including endothelial
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Fig. 5. Overexpression of RBP4 in mice impaired mitochondrial dynamic and promoted apoptosis in vivo. (A, B) Representative Western blot and quantitative analysis of mitochondrial fusion and fission proteins in aortas of C57BL/6J and RBP4-Tg mice. (C, D) Representative Western blot and quantitative analysis of apoptosis-related proteins in aortas of C57BL/6J and RBP4-Tg mice. (EeH) Representative Western blot and quantitative analysis of phosphorylation of PI3K and Akt. Values were presented as means ± SEM, n ¼ 10 each group *p < 0.05 vs. control (C57BL/6J), **p < 0.01 vs. control (C57BL/6J).
cells [39,40]. Consistent with the notion that release of cytochrome C from the mitochondria is a central event in the apoptotic process [41], an increase in cytochrome C release from the mitochondria was observed in RBP4-treated HAECs and arteries from RBP4-Tg
mice, indicating an enhanced apoptosis. Members of the Bcl-2 family play important roles in the regulation of mitochondriainitiated apoptosis which directly controls the mitochondrial membrane permeability and subsequently regulates the release of
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apoptotic factors into the cytoplasm [42]. Bax is a pro-apoptotic protein belonging to the Bcl-2 family, predominantly in the cytosol in an inactive form. In case of apoptosis, Bax assumes an active conformation, thus allowing the translocation from cytosol to mitochondria, and a cascade of events that lead to apoptosis with caspase 3 as the final executor [43]. Our data demonstrated that either in RBP4-treated HAECs or RBP4-Tg mice, Bax were significantly upregulated, while Bcl-2 was largely reduced, leading to acceleration of apoptosis. To our knowledge, this is the first report to link RBP4 with endothelial mitochondrial dysfunction and apoptosis both in vitro and in vivo. PI3K/Akt signals is reported to exert an antiapoptotic action through Bax, Bcl-2 [44e48], therefore, we went on to examine whether the apoptosis induced by RBP4 was dependent on PI3K/ Akt pathway. The serine/threonine kinase Akt is one of the major downstream targets of PI3K. At the membrane, Akt is phosphorylated at two key residues: Thr308 of the activation loop by PDK1 and Ser473 in the hydrophobic motif of the C-terminal tail. Maximal AKT activity is dependent on the phosphorylation status of both Thr308 and Ser473 residues. Furthermore, phosphorylation at Ser473 precedes the phosphorylation of Thr308 and is critical for the recognition and activation of Akt by PDK1. Our study showed that RBP4 led to a significant reduction of Akt phosphorylation at Ser473 but no obvious effect on the phosphorylation level at Thr308 both in vitro and in vivo. Altogether, our results imply that RBP4-induced endothelial apoptosis is PI3K/Akt dependent. Previous reports have indicated that altered mitochondrial dynamics serve as a major cause of mitochondrial dysfunction. The mitochondria constantly undergo fusion and fission processes, which are critical for the release of cytochrome C and are associated with cellular apoptosis signaling [49]. We further examined whether RBP4 affected mitochondrial morphology and integrity by disrupting the homeostasis of fusion and fission. Our findings demonstrated that the expression of mitochondrial fusion protein Mfn1 was lower in the aorta of RBP4-Tg mice and RBP4-stimulated HAECs. Conversely, RBP4 incubation and overexpression in mice resulted in a significant increase in the expression of mitochondrial fission proteins Fis1 and Drp-1. These findings suggested that RBP4induced alterations in mitochondrial dynamics impaired endothelial cell function. However, the underlying mechanism for this observation is unknown and warrants further study. Taken together, the findings of this study reveal a novel mechanism through which RBP4 exacerbates endothelial mitochondrial function and apoptosis by promoting mitochondrial oxidative stress via the regulation of mitochondrial fusion and fission process. Our findings shed new light on the role of RBP4 far beyond an adipokine related to insulin resistance, and proved that it may be a potential therapeutic target for preventing the progression of cardiovascular disease.
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Funding
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This work was supported by grants from the National Natural Science Foundation of China (81172663).
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Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Retinol binding protein 4 induces mitochondrial dysfunction and vascular oxidative damage Jingjing Wang a, b, 1, Hongen Chen a, b, 1, Yan Liu a, b, Wenjing Zhou b, Ruifang Sun b, Min Xia a, b, * a b
Guangdong Provincial Key Laboratory of Food, Nutrition and Health, China Department of Nutrition, School of Public Health, Sun Yat-sen University (Northern Campus), Guangzhou, Guangdong Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 November 2014 Received in revised form 8 March 2015 Accepted 25 March 2015 Available online 28 March 2015
Objectives: Mitochondrial dysfunction has been implicated in cardiovascular diseases. Elevation of serum retinol binding protein 4 (RBP4) in patients has been linked to cardiovascular disease. However, the role of RBP4 on mitochondrial oxidative stress and vascular oxidative damage is not well demonstrated. Therefore, we evaluated the impact of RBP4 on the mitochondrial reactive oxygen species (ROS) and dynamics in the pathogenesis of cardiovascular diseases. Methods and results: RBP4 treatment increased mitochondrial superoxide generation in a dosedependent manner in human aortic endothelial cells (HAECs). Exposure to RBP4 also promoted mitochondrial dysfunction as determined by decreased mitochondrial content and integrity as well as membrane potential in HAECs. Incubation with RBP4 suppressed mitofusin (Mfn)-1 protein expression, but enhanced dynamin-related protein-1 (Drp1) and fission-1 (Fis1) protein expression in HAECs, suggesting an impairment of mitochondrial fusion and fission dynamics. Moreover, RBP4 treatment significantly induced endothelial apoptosis, increased the expression of Cytochrome C and Bax, but decreased the expression of Bcl-2. Furthermore, RBP4 stimulation suppressed phosphatidyl inositol 3kinase (PI3K)/Akt signaling in HAECs. Finally, RBP4-Tg mice exhibited severe mitochondrial dysfunction and vascular oxidative damage in aorta compared with wide-type C57BL/6J mice. Conclusion: The present study uncovers a novel mechanism through which RBP4 induces vascular oxidative damage and accelerates the development of atherosclerosis. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Retinol binding protein 4 Mitochondrial dysfunction ROS Endothelial cells
1. Introduction Mitochondrial dysfunction including increased production of reactive oxygen species (ROS) in mitochondria, accumulation of mitochondrial DNA damage, and progressive respiratory chain dysfunction contributes to the pathogenesis of atherosclerosis in human investigations and animal models of oxidative stress [1,2]. There is growing appreciation of the importance of altered mitochondrial dynamics in cardiovascular disease [3,4]. Mitochondria undergo cycles of fusion to form networks and fission to form smaller individual mitochondria [5,6]. Proteins controlling fusion include mitofusin (Mfn)-1, Mfn2, and optic atropy-1 (Opa1). Fission
* Corresponding author. Department of Nutrition, School of Public Health, Sun Yat-sen University (Northern Campus), Guangzhou, Guangdong Province, China. E-mail address: [email protected] (M. Xia). 1 Jingjing Wang and Hongen Chen contribute equally to this paper. http://dx.doi.org/10.1016/j.atherosclerosis.2015.03.036 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
is regulated by dynamin-related protein-1 (Drp1) and fission-1 (Fis1). Fusion may be beneficial by allowing the distribution of metabolites, proteins, and DNA throughout the network [7]. At the end of their life cycle, dysfunctional mitochondria and damaged mitochondrial components are eliminated by fission and subsequent autophagy [8]. Under pathological conditions, including atherosclerosis, fission is increased and autophagy is impaired, leading to a loss of mitochondrial networks, accumulation of small dysfunctional mitochondria, and increase of mitochondrial ROS [9]. Retinol-binding protein 4 (RBP4), an adipokine primarily secreted by adipocytes and hepatocytes, is the sole carrier of retinol in the blood, responsible for the transportation of retinol from liver stores to peripheral tissues [10,11]. The potential link between RBP4 and insulin resistance and diabetes had been suggested by several reports showing that the RBP4 levels were elevated in insulinresistant mice and in humans with obesity and type 2 diabetes mellitus [12e14]. Recent clinical studies have linked elevated
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2. Materials and methods
peroxidation was determined by the measurement of thiobarbituric acid reactive substances (TBARS) in arteries. The freshly obtained arteries were grinded with liquid nitrogen, lysed and then 500 ml of homogenate was mixed with thiobarbituric acid and heated in boiling water for 40 min. When cooling down, the absorbance was measured at 532 nm (TECAN, Sunrise). The assay of total superoxide dismutase (SOD) activity was based on the capacity of SOD to inhibit the oxidation of WST-1 2-(4iodophenyl)-3-(4-nitrophenyl) -5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt. Activity of Cu,Zn-SOD isoform was measured by adding the inactivator of Mn-SOD and then Mn-SOD activity was calculated by subtracting Cu, Zn-SOD activity from the total SOD activity. The absorbance was measured at 550 nm (TECAN, Sunrise).
2.1. Materials
2.6. Mitochondrial membrane potential (DJm)
Recombinant human RBP4 (NP_006735.2, Met 1-Leu 201) expressed in HEK293 cells with a C-terminal polyhistidine tag was obtained from Sino Biological Inc. (Beijing, China). Endothelial Cell Medium (ECM), fetal bovine serum, endothelial cell growth supplement (ECGS) and antibiotic mixtures (Penicillin-Streptomycin) were provided by American ScienCell Technologies Inc (Carlsbad, USA).
After incubation with RBP4 for 24 h, HAECs were washed with PBS, incubated with JC-1 dye (5,5,6,6-tetrachloro-1,1,3,3tetraethylbenzimidazolyl-carbocyanine iodide) (5 mg/mL, Enzo, Farmingdale, New York, USA) for 10 min at 37 C and then observed with a confocal microscope (Leica TCS SP5 II). According to the manufacturer's instructions, two fluorescences could be captured in live cells. An orange J-aggregate emission profile was detected in healthy cells, and as membrane potential decreases, JC-1 monomers were generated, resulting in a shift to green emission. For the mitochondrial membrane potential of C57BL/6J and RBP4-Tg mice, arteries from the two groups were measured using an assay kit (GMS10013.4, GENMED, Shanghai, China). Freshly obtained arteries were washed twice with GENMED cleaner, incubated with GENMED staining at 37 C for 60 min and then lysed with GENMED lysate before images were captured by confocal microscope at 490/530 nm. According to the instructions, only green flourescence could be detected in tissues.
serum RBP4 specifically to cardiovascular disease, including hypertension [15], stroke [16], and atherosclerosis [17]. Notably, the association between higher circulating RBP4 levels and an increased risk of coronary heart disease (CHD) was confirmed among women in the Nurses' Health Study [18], indicating that RBP4 may contribute to the development of atherosclerosis. The finding that elevation of RBP4 is correlated with cardiovascular disease warrants our investigation into the possible role of RBP4 in the pathogenesis of vascular disease. The present study was designed to investigate the effect of RBP4 on mitochondrial dynamics and vascular oxidative damage in vitro and in vivo.
2.2. Experimental animals Transgenic mice expressing human RBP4 (RBP4-Tg, Stock Number: 031125- UUCD) were provided by the Mutant Mouse Regional Resource Centers (MMRRC, Rockville, North America). C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The animals (n ¼ 10 each group) were fed with a regular chow diet (Medicience Ltd, Yangzhou, China) for two months before applying for experiments (at 16 weeks of age). The animals were housed in a specific pathogenefree facility in a temperature-controlled room (22e24 C) with a 12 h lightedark cycle and given free access to food and water. The approval for this animal research was granted by the ethic review board of Sun YatSen University. 2.3. Cell culture Human aortic endothelial cells (HAECs) were obtained from ScienCell Research Laboratories and cultured with the ECM supplemented with FBS (10% v/v), penicillin (100 U/mL), streptomycin (100 mg/mL), heparin (90 mg/mL), and endothelial cell growth supplement (20 mg/mL) at 37 C under 5% CO2 atmosphere. Only three to ten passages of HAECs were used in these experiments. 2.4. Measurement of mitochondrial ROS production Cells were precultured with RBP4 for 24 h, washed with PBS, and then, stained with MitoSOX™ Red (5 mmol/L, invitrogen, Oregon, USA), a mitochondrial superoxide indicator for 10 min at 37 C, protected from light. Cells were washed gently three times with warm PBS, and then, stained with Hoechst 33258 (2 mg/mL, H21491, Invitrogen, Oregon, USA) for another 20 min at 37 C. The fluorescence of MitoSOX™ Red was detected under a confocal microscope (Leica TCS SP5Ⅱ) [19]. 2.5. Detection of SOD and MDA Superoxide dismutase (SOD) activity and MDA levels were measured using assay kits (Jiancheng Biotechnology Co. Nanjing, China) according to the manufacturers' instructions. The lipid
2.7. Mitochondrial quantification and fragmentation Cells were treated with RBP4 for 24 h, washed with PBS, then incubated with MitoTracker® Green FM (50 nM; invitrogen, Oregon, USA) for 20 min at 37 C. Cells were washed gently three times with warm PBS, and then, stained with Hoechst 33258 (2 mg/mL; H21491, Invitrogen, Oregon, USA) for another 20 min at 37 C. The fluorescence of MitoTracker® Green FM was detected under a confocal microscope (Leica TCS SP5Ⅱ) and the fluorescence intensity was quantified by Image J software. 2.8. Transmission electron microscopy A stretch of thoracic aorta was immediately removed, fixed in a solution of 2.5% glutaraldehyde, postfixed in 1% osmium teroxide for 1~2 h, and then dehydrated in gradient ethanol. The ultrathin slices were viewed with a Tecnai G2 Spirit Twin electron microscope (American FEI, Czech). 2.9. Flow cytometry Quantification of mitochondrial ROS and content in HAECs were performed using Mito-SOX and MitoTracker® Green respectively. Aliquots of 106~108 cells were centrifuged to obtain a cell pellet. Resuspending the cells gently in pre-warmed (37 C) staining solution with the fluorescent probes as described above in the dark at 37 C. After staining, the cells were re-pelleted by centrifugation and resuspended in fresh PBS, and then, a flow cytometric analysis was conducted. All analyses were performed by FACScan (BD, FACS Calibur) and the FlowJo V7.6.5 software.
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2.10. Detection of apoptosis To identify chromatin condensation, HAECs were cultured with Hoechst 33258 (2 mg/mL, H21491, invitrogen, Oregon, USA) for 30 min at 37 C. The blue fluorescent was efficiently excited with a UV laser and the stained cells were visualized using a confocal microscope at a magnification of 400. For quantification, a blind analysis from three to four randomized fields was performed. Cells were counted as apoptotic when the stained nucleus produced bright blue fluorescence due to chromatin condensation [20]. 2.11. Western blot Total protein samples were extracted from the aorta or HAECs using the RIPA lysis buffer. Aliquots (30 mg) were separated by SDSPAGE and then transferred to a PVDF membrane. After blocking with 5% BSA, the blots were incubated with various primary antibodies followed by horseradish peroxidase-labeled secondary antibodies. The protein bands were detected with a SuperSignal West Pico Chemiluminescent kit (Thermo, Massachusetts, USA). Primary antibodies for Mfn1, Mfn2, Opa-1, Drp-1 Fis-1, and GAPDH were from Santa Cruz (Texas, USA) and PI3K, phospho-PI3K, AKT, phospho-AKT (Ser473), phospho-AKT (Thr308), Cytochrome C, Bcl2 and Bax were from Cell Signaling (Boston, USA). 2.12. Statistical analysis Statistical analyses were performed with the SPSS 13.0. Data were expressed as mean ± S.E.M. from five or more independent experiments run in triplicate. All variables were tested by Shapiro-
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Wilk test for normal distribution. Student's unpaired t test was used for comparison between two groups. When more than two groups were compared One-way analysis of variance (ANOVA) was used. P < 0.05 was considered as statistically significant. 3. Results 3.1. Effect of RBP4 on the mitochondrial superoxide generation We applied the fluorescent probe MitoSOX™ Red, a superoxidesensitive fluorescent dye, to determine the effect of RBP4 on mitochondrial accumulation of superoxide by using confocal microscopy. Compared with untreated control, RBP4 treatment caused a dose-dependent increase of bright red fluorescence, indicating an enhanced mitochondrial superoxide production (Fig. 1A and B). This result was further confirmed by flow cytometry analysis, showing that RBP4 (40 mg/mL) exposure resulted in about 2.6-fold increase of fluorescence intensity compared with untreated control (Fig. 1C and D). 3.2. Effect of RBP4 on mitochondrial dysfunction in endothelial cells To investigate the effect of RBP4 on mitochondrial dysfunction, we used MitoTracker Green, a specific marker for mitochondria, to ascertain mitochondrial quantity and integrity. RBP4 exposure resulted in a concentration-dependent reduction of the green fluorescence (Fig. 2A and B), suggesting a significant decrease of mitochondrial content. This finding was further confirmed by the flow cytometry analysis, demonstrating about ~38% decrease of mitochondrial content in response to RBP4 treatment (Fig. 2C and
Fig. 1. RBP4 induced mitochondrial ROS formation. HAECs were cultured to 80% and treated with different doses of RBP4. Mitochondrial ROS production was measured by MitoSOX510/580nm and fluorescence was measured by (A) confocal microscope (magnification of 400) and (C) flow cytometry. (B) Quantitative analysis of fluorescence intensity. (D) Mean integrated fluorescence (MIFL) for Mito-SOX for each condition in (C). Results were represented as mean ± SEM of five individual experiments;*p < 0.05 vs. control (0 mg/mL), ** p < 0.01 vs. control (0 mg/mL).
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D). The proportion of fragmented versus tubular organelles was augmented in response to RBP4 treatment in a dose-dependent fashion (Fig. 2E). Mitochondrial membrane potential (DJm) is essential for normal mitochondrial function. Loss of DJm caused mitochondrial membrane depolarization and triggered a cascade of mitochondrion-dependent apoptotic signaling [21,22]. We therefore determined the effect of RBP4 on this mitochondrial parameter using JC-1 staining. Treatment of HAECs with RBP4 depolarized DJ (red fluorescence/green fluorescence) in a dose-dependent manner after 24 h incubation (Fig. 2F and G). 3.3. Effect of RBP4 on mitochondrial dynamic in endothelial cells Mitochondrial fusion and fission balance controlled the number, length and tubular shape of mitochondria [23]. Once the balance was broken, mitochondrial networks were damaged leading to mitochondrial dysfunction [24]. We thus determined the effect of RBP4 on mitochondrial fusion (Mfn1, Mfn2, Opa1) and fission (Drp1 and Fis1) proteins, which are key regulators of mitochondrial morphology. RBP4 treatment decreased the Mfn1 protein expression in a dose-dependent manner in HAECs. On the contrary, RBP4 stimulation increased the protein expression of both Drp1 and Fis1 (Fig. 2H and I). 3.4. Effect of RBP4-mediated mitochondrial dysfunction on endothelial apoptosis Mitochondrial dysfunction plays a crucial role in the regulation of the endogenous pathways of apoptosis activated by oxidative stress [25]. To determine whether RBP4- induced mitochondrial dysfunction was associated with apoptosis, we used Hoechst 33258 staining to analyze chromatin condensation, a key hallmark of apoptosis. Compared with control, RBP4 (20 mg/mL) treatment caused approximate 1.3-fold increase in apoptotic rate, as presented with intense fluorescence staining. Exposure to RBP4 treatment (40 and 60 mg/mL) induced a 2~2.5-fold increase in apoptosis cells compared to the control (Fig. 3A and B, P < 0.01). Furthermore, the apoptosis-related proteins were assessed. RBP4 treatment significantly increased the expression of Cytochrome C and Bax, but largely decreased the expression of Bcl-2 (Fig. 3C and D). 3.5. Effect of RBP4 on PI3K/AKT signaling pathway in endothelial cells It has been demonstrated that the activation of PI3K/AKT signaling pathway inhibits cellular apoptosis and promotes cell survival [26]. To determine whether RBP4 had an effect on insulin signaling in HAECs, the phosphorylation of AKT, the downstream effector of PI3K, was examined in RBP4-treated HAECs as described above. Dose-response studies demonstrated that compared to lowdose RBP4 treatment, PI3K phosphorylation was significantly decreased after treatment with high-dose RBP4. The phosphorylation of AKT at Ser473 sites was significantly decreased, while, the phosphorylation of AKT at Thr308 site had no obvious change. Total AKT and PI3K expression was not changed by any dose of RBP4 used
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in these studies (Fig. 3EeH). 3.6. Overexpression of RBP4 induces mitochondrial dysfunction and vascular oxidative damage in vivo To gain insight into the role of RBP4 in mitochondrial dysfunction and oxidative damage in vivo, we next determined its metabolic effects in the arteries of RBP4-Tg mice. The MDA production of RBP4-Tg mice arteries were ~1.6-fold higher than that of C57BL/6J (Fig. 4A, P < 0.05). Superoxide dismutases (SOD) are the major antioxidant enzymes for superoxide removal, and in mammals, three SOD isoforms are expressed: copper/zinc SOD (CuZn-SOD, SOD1), located in the cytoplasm; manganese SOD (Mn-SOD, SOD2), localized in the mitochondrial matrix; and extracellular SOD (ECSOD, SOD3). The total SOD activity from arteries of RBP4-Tg mice decreased ~20% compared with C57BL/6J (Fig. 4B, P < 0.05). MnSOD activity from mitochondria of RBP4-Tg arteries decreased ~42% compared with C57BL/6J (Fig. 4C, P < 0.05), however, CuZnSOD activity did not reduced significantly (Fig. 4D), implying that reduced antioxidant capacity was mainly attributed to the dysfunction of mitochondria. Analysis of the morphology of mitochondria by transmission electron microscope revealed a vague or absent mitochondrial crest and mitochondrial division section in RBP4-Tg mice artery (Fig. 4E). In addition, JC-1 monomers (green) were significantly highly generated in RBP4-Tg mice arteries compared with C57BL/6J (Fig. 4F and G), indicating an aggravated mitochondrial membrane depolarization in RBP4-Tg mice. RBP4-Tg mice had a lower level of mitochondrial fusion protein expression of Mfn1 than that in C57BL/6J mice (P < 0.05). Conversely, a higher level of mitochondrial fission proteins, including Drp1 and Fis-1 was observed in RBP4-Tg mice compared with C57BL/6J mice (Fig. 5A and B, P < 0.05). Furthermore, arteries from RBP4-Tg mice had a lower level of Bcl-2 and a higher level of Cytochrome C and Bax compared with C57BL/6J (Fig. 5C and D). In consistence with decreased PI3K/Akt activity in HAECs, phosphorylation of PI3K and Akt at Ser473 were significantly attenuated in arteries from RBP4-Tg mice, while phosphorylation of AKT at Thr308 site had no obvious change (Fig. 5EeH). 4. Discussion In the present study, we presented a novel mechanism by which RBP4 deteriorates endothelial mitochondrial function and promotes vascular oxidative stress. Both exogenous RBP4 treatment on HAECs and overexpression of RBP4 in mice induced mitochondrial superoxide production, impaired mitochondrial content and integrity as well as membrane potential, leading to endothelial mitochondrial dysfunction. The loss of function was due to the imbalance of fusion and fission cycle in mitochondria, with reduced Mfn1 and significantly increased Drp1 and Fis-1 expressions. RBP4induced mitochondrial dysfunction was also accompanied by apoptosis in both HAECs and RBP4-Tg mice, which was associated with impaired PI3K/AKT signaling pathway. A substantial number of studies have revealed that among the potential causes of cardiovascular diseases, oxidative stress mediated changes within the cardiovascular environment are essential
Fig. 2. RBP4 promoted mitochondrial dysfunction. Mitochondrial content was measured by MitoTracker Green and fluorescence was measured by (A) confocal microscope (magnification of 400) and (C) flow cytometry. (B) Quantitative analysis of fluorescence intensity. (D)Mean integrated fluorescence (MIFL) for MitoTracker Green for each condition in (C). (E) A shift of mitochondrial morphology from tubular to fragmented forms was observed in response to high-dose RBP4 stimulation (magnification of 1000 top and 10,000 down). (F) Reduced mitochondrial membrane potential was measured with JC-1. (G) Quantitative analysis of the ratio of JC-1 oligomer (red)/monomer (green) fluorescence. (H) Representative Western blot analysis of mitochondrial fusion and fission proteins. (I) Quantitative analysis of the protein levels. Results were represented as mean ± SEM of five individual experiments; *p < 0.05 vs. control (0 mg/mL), **p < 0.01 vs. control (0 mg/mL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Effect of RBP4-mediated mitochondrial dysfunction on endothelial apoptosis. Cultured HAECs were incubated with RBP4 for 24 h. (A) Apoptotic HAECs were calculated by Hoechst 33258346/460nm staining. Original magnification 400. (B) Apoptotic cells were counted based on morphology, and the number of apoptotic cells was expressed as percentage of total cells. (C, D) Representative Western blot and quantitative analysis of Cytochrome C, Bax, Bcl-2 protein expressions. (E) Representative Western blot analysis of PI3K, p-PI3K, AKT, p-AKTser473, p-AKTThr308 protein expressions in HAECs. (FeH) Quantitative analysis of PI3K and AKT phosphorylation. Results were represented as mean ± SEM of five individual experiments; *p < 0.05 vs. control (0 mg/mL), **p < 0.01 vs. control (0 mg/mL).
in the progression of cardiovascular diseases [27e29]. Although ROS is capable of targeting a variety of subcellular components, the mitochondrial membranes, proteins and mtDNA appear particularly sensitive to oxidative stress [30]. Increasing evidence indicates
that mitochondrial oxidative stress and the subsequently induced mitochondrial dysfunction contribute to the multiple pathological processes underlying the cardiovascular diseases. Mitochondrial integrity and functions are essential in determining individual
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Fig. 4. Overexpression of RBP4 induced vascular oxidative damage in mice. Freshly obtained arteries from C57BL/6J and RBP4-Tg mice were measured to determine (A) the TBARs levels and (BeC) the total SOD activity, Mn-SOD isoform from mitochondria and CuZn-SOD activity. (E) The ultrastructure of mitochondria in arteries of C57BL/6J and RBP4-Tg mice were observed using a transmission electron microscope (Original magnification 46,000). (F) Freshly obtained arteries of C57BL/6J and RBP4-Tg mice were stained with GENMED staining and observed at 490 nm excitation and 530 nm emission. (G) Quantitative analysis of green fluorescence intensity. Values were presented as means ± SEM, n ¼ 10 each group; *p < 0.05 vs. control (C57BL/6J), **p < 0.01 vs. control (C57BL/6J).
vulnerability to common CVD risk factors. Besides, mitochondrial damage and dysfunction have been observed in both human cells and animal models [31e33]. The link between elevation of serum RBP4 and cardiovascular diseases had been reported by several clinical studies [34e37], however the exact role of RBP4 in the pathogenesis of cardiovascular disease remains unknown. Consistent with the report that incubation with holo-RBP4 dose-dependently increases the expression of Nox2 and Nox4, leading to activation of NADPH oxidases and increase in mitochondrial O2$ [38], in the present study, we found that at cellular levels RBP4
treatment dose-dependently induced mitochondrial ROS, impaired mitochondrial content and integrity and decreased the membrane potential. A similar observation was evidenced in RBP4-Tg mice, with a higher expression of lipid peroxidation, abnormal mitochondria ultrastructure, and reduced mitochondrial antioxidant capacity in the artery. As a consequence of mitochondrial dysfunction, the negative alterations including impaired ATP production and increased ROS generation could affect the physiology and promote apoptosis in all cell types involved in cardiovascular disease including endothelial
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Fig. 5. Overexpression of RBP4 in mice impaired mitochondrial dynamic and promoted apoptosis in vivo. (A, B) Representative Western blot and quantitative analysis of mitochondrial fusion and fission proteins in aortas of C57BL/6J and RBP4-Tg mice. (C, D) Representative Western blot and quantitative analysis of apoptosis-related proteins in aortas of C57BL/6J and RBP4-Tg mice. (EeH) Representative Western blot and quantitative analysis of phosphorylation of PI3K and Akt. Values were presented as means ± SEM, n ¼ 10 each group *p < 0.05 vs. control (C57BL/6J), **p < 0.01 vs. control (C57BL/6J).
cells [39,40]. Consistent with the notion that release of cytochrome C from the mitochondria is a central event in the apoptotic process [41], an increase in cytochrome C release from the mitochondria was observed in RBP4-treated HAECs and arteries from RBP4-Tg
mice, indicating an enhanced apoptosis. Members of the Bcl-2 family play important roles in the regulation of mitochondriainitiated apoptosis which directly controls the mitochondrial membrane permeability and subsequently regulates the release of
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apoptotic factors into the cytoplasm [42]. Bax is a pro-apoptotic protein belonging to the Bcl-2 family, predominantly in the cytosol in an inactive form. In case of apoptosis, Bax assumes an active conformation, thus allowing the translocation from cytosol to mitochondria, and a cascade of events that lead to apoptosis with caspase 3 as the final executor [43]. Our data demonstrated that either in RBP4-treated HAECs or RBP4-Tg mice, Bax were significantly upregulated, while Bcl-2 was largely reduced, leading to acceleration of apoptosis. To our knowledge, this is the first report to link RBP4 with endothelial mitochondrial dysfunction and apoptosis both in vitro and in vivo. PI3K/Akt signals is reported to exert an antiapoptotic action through Bax, Bcl-2 [44e48], therefore, we went on to examine whether the apoptosis induced by RBP4 was dependent on PI3K/ Akt pathway. The serine/threonine kinase Akt is one of the major downstream targets of PI3K. At the membrane, Akt is phosphorylated at two key residues: Thr308 of the activation loop by PDK1 and Ser473 in the hydrophobic motif of the C-terminal tail. Maximal AKT activity is dependent on the phosphorylation status of both Thr308 and Ser473 residues. Furthermore, phosphorylation at Ser473 precedes the phosphorylation of Thr308 and is critical for the recognition and activation of Akt by PDK1. Our study showed that RBP4 led to a significant reduction of Akt phosphorylation at Ser473 but no obvious effect on the phosphorylation level at Thr308 both in vitro and in vivo. Altogether, our results imply that RBP4-induced endothelial apoptosis is PI3K/Akt dependent. Previous reports have indicated that altered mitochondrial dynamics serve as a major cause of mitochondrial dysfunction. The mitochondria constantly undergo fusion and fission processes, which are critical for the release of cytochrome C and are associated with cellular apoptosis signaling [49]. We further examined whether RBP4 affected mitochondrial morphology and integrity by disrupting the homeostasis of fusion and fission. Our findings demonstrated that the expression of mitochondrial fusion protein Mfn1 was lower in the aorta of RBP4-Tg mice and RBP4-stimulated HAECs. Conversely, RBP4 incubation and overexpression in mice resulted in a significant increase in the expression of mitochondrial fission proteins Fis1 and Drp-1. These findings suggested that RBP4induced alterations in mitochondrial dynamics impaired endothelial cell function. However, the underlying mechanism for this observation is unknown and warrants further study. Taken together, the findings of this study reveal a novel mechanism through which RBP4 exacerbates endothelial mitochondrial function and apoptosis by promoting mitochondrial oxidative stress via the regulation of mitochondrial fusion and fission process. Our findings shed new light on the role of RBP4 far beyond an adipokine related to insulin resistance, and proved that it may be a potential therapeutic target for preventing the progression of cardiovascular disease.
[6] [7] [8]
[9]
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[12]
[13]
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[15] [16]
[17]
[18]
[19]
[20]
[21]
[22]
[23] [24] [25] [26] [27]
[28] [29]
Funding
[30] [31]
This work was supported by grants from the National Natural Science Foundation of China (81172663).
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