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PEG-coated gold nanoparticles attenuate β-adrenergic recepto r mediated-cardiac hypertrophy
作者:李子健[1] 
单位:北京大学第三医院[1]  
文章号:W137890  
2019/8/21 18:02:00    
文字大小:

PEG-coated gold nanoparticles attenuate β-adrenergic receptor mediated-cardiac hypertrophy
Yuhui Qiaoa, Baoling Zhua, Aiju Tiana, Zijian Lia*


  a Department of Cardiology, Institute of Vascular Medicine, Peking University Third Hospital, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Ministry of Health, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education and Beijing Key Laboratory of Cardiovasicular Receptors Research Beijing 100191, China.


  *Correspondence author(email: lizijian@bjmu.edu.cn)

 

Abstract


  Gold-(Au-) nanoparticles (AuNPs) are widely used as drug delivery vehicle, which can accumulate in the heart through the blood circulation. Therefore, it is very important to understand the effect of AuNPs on heart, especially under pathological condition. In this study, we found PEG-coated gold nanoparticles attenuate β-adrenergic receptor mediated-acute cardiac hypertrophy and inflammation. However, both isoproterenol (ISO), a non-selective β-AR agonist, and AuNPs did not induce cardiac function change or cardiac fibrosis. AuNPs exerted anti-cardiac hypertrophy effect by decreasing β1-AR expression and its downstream ERK1/2 hypertrophic pathway. Our results indicated that AuNPs might be safe and have the potential to be used as multiple-functional materials (drug carrier systems and anti-cardiac hypertrophy agents).

 

Key words

  AuNPs; Cardiac hypertrophy; β-adrenergic receptor; ERK1/2 signalling pathway

 

Abbreviations
ISO                 Isoprenaline
β-adrenergic receptor   β-AR
LVPW;d             Diastolic left ventricular posterior wall thickness
LVID;d              Left ventricular end-diastolic inner-diameter
LVID;s              Left ventricular end-systolic inner-diameter
EF                  Ejection fraction    
FS                  Fractional shortening
HW                 Heart weight
TL                  Tibia length

 

1.Background


  Along with the advances in nanotechnology, nanoparticles have been applied widely in biomedicine [1,2]. In particular, gold nanoparticles (AuNPs) are a potential candidate for the development of dignositic and therapeutic methods, due to their unique physical, chemical, optical and pharmacological properties. For example, gold cores are inert, stable, biocompatible and low-toxic. In addition, AuNPs are easy to  prepare and functionalize [3,4]. Therefore, AuNPs have great potential for many biomedical applications, such as drug or protein delivery, gene transfection, cancer therapy, biomedical imaging, bio-labeling and molecular diagnostics tools. Furthermore, the surface of AuNPs can be modificated to improve specifity and safety of its application in clinical or research [5,6]. Since AuNPs have shown great potential for the widely application in diagnosis and treatment of diseases, it is important to evaluate the safety of AuNPs in the dieases. 

  Cardiac diseases have been the leading cause of death worldwide [7]. AuNPs are widely used in the diagnosis and treatment of cardiac dieases [8-11]. However, there have been only a few studies that examined the effect of AuNPs on hearts. So it is necessary to investigate the safety of AuNPs for hearts under both physiological and pathological conditions. Our previous study has proved the safety of AuNPs under physiological condition [12], so we focused on the safety of AuNPs for hearts under pathological condition in the present study.


  Cardiac hypertrophy is the important pathological basis for various heart diseases and is an independent risk factor for morbidity and mortality of heart failure [13]. While the safety of AuNPs in the condition of cardiac hypertrophy is still unclear. A key factor of cardiac hypertrophy is the over-activation of β-adrenergic receptor (β-AR) [14] and β-blockers have been one of the standardized therapeutic drugs for heart failure in clinical [15]. Thus isoprenaline (ISO), the agonist of β-AR, has been widely used to establish the animal model of cardiac hypertrophy [16,17]. ISO is reported to induce both chronic and acute cardiac hypertrophy in animal experiments. The chronic cardiac hypertrophic model is to simulate the chronic progression of heart remodeling in some chronic cardiovascular diseases such as hypertension [18]. The acute cardiac hypertrophic model reflect cardiac remodeling following acute cardiac injuries [19]. The safety of AuNPs in ISO-induced chronic cardiac hypertrophic model has been demonstrated in our previous study [20]. Thus in the present study, the safety of AuNPs in ISO-induced acute cardiac hypertrophic model was investigated.


  The results in this study indicated that the accumulation of AuNPs in heart depends on their sizes and the accumulation of AuNPs in heart attenuate β-AR-mediated acute cardiac hypertrophy and inflammation through decrease of β1-AR expression and its downstream ERK1/2 hypertrophy signaling. These novel findings will be helpful for the wider applications of AuNPs in cardiac diseases.

 

2. Methods


  2.1. Characterization of the AuNPs
  Three different sizes (13, 30 and 50 nm) of PEG-coated gold nanoparticles (AuNPs) used in this experiment were from Nanocs Inc., New York (http://www.nanocs.com). The morphology size and aggregation state of the AuNPs were evaluated using transmission electron microscopy (TEM, JEM-200CX, Jeol Ltd., Japan) and Multiskan GO (Thermo Scientific, Ltdl, USA). The PEG-coated AuNPs suspension was sonicated for 5 min before use.


  2.2. Establishment of animal model
  Our investigation was approved by the Biomedical Research Ethics Committee of Peking University (LA 2010-048) and strictly adhered to the American Physiological Society’s “Guiding Principles in the Care and Use of Vertebrate Animals in Research and Training”. 10-12 weeks male FVB/N mice were from laboratory animal department of Peking University Health Science Center. Mice were housed in groups of four and maintained on a 12 h dark/light cycle in a room with controlled temperature (25 ± 2 ◦C). Mice had free access to food and water. Cardiac hypertrophy model was established by subcutaneous injection of ISO (200 mg/kg/day, dissolved in saline, Sigma Aldrich, St. Louis, USA) for 3 consecutive days.

 

  There are five groups in total (eight to ten mice each group): (i) control group: daily administration of saline for 3 days; (ii) ISO group: daily subcutaneous administration of ISO (200 mg/kg/day) for 3 days. (iii) ISO+13nm AuNPs group: daily subcutaneous administration of ISO (200 mg/kg/day) for 3 days. 540 μg/kg AuNPs of 13nm were injected into the tail vein on the same days after ISO injections. (iv) ISO + 30nm AuNPs group: daily subcutaneous administration of ISO (200 mg/kg/day) for 3 days. 540 μg/kg AuNPs of 30nm were injected into the tail vein on the same days after ISO injections. (v) ISO+50nm AuNPs group: daily subcutaneous administration of ISO (200 mg/kg/day) for 3 days. 540 μg/kg AuNPs of 50nm were injected into the tail vein on the same days after ISO injections. The contents of AuNPs of all the three sizes (13, 30 and 50nm) are all 0.01% Ag/ml. The volume of injection was adjusted to 5μl/g of mice weight. The PEGylated AuNPs suspension was sonicated for 5 min before use to make the AuNPs disperse adequately. The treatment of animals in each group is shown in Table 1.


  2.3. Echocardiographic analysis
  Echocardiography analysis was performed one day after the last injection. Mice were anaesthetized with 1.5% isoflurane (Baxter Healthcare Corporation, New Providence, USA). Echocardiographic images were obtained by the Visualsonics high-resolution Vevo 770 system (VisualSonics, Inc. Canada). Two-dimensional short-axis views were obtained at the level of the papillary muscle. The diastolic left ventricular posterior wall thickness (LVPW;d) and systolic left ventricular posterior wall thickness (LVPW;s) were measured to calculate the ejection fraction (EF) and fractional shortening (FS). All measurements were averaged from three consecutive cardiac cycles. Cardiac systolic function was represented by the values of EF and FS. Doppler echocardiograms were captured via an apical four chamber view. Transmitral flow Doppler was obtained through mitral flow center and two characteristic E wave and A wave were obtained. Tissue Doppler images were obtained through the mitral annulus and Doppler velocities E´ and A´ were obtained. E/A , E´/A´ and E/E’ ratios were calculated to evaluate cardiac diastolic function. The average of three consecutive cardiac cycles were taken for each parameter. Echocardiography procedures were operated in accordance with the guideline of American Society of Echocardiography.


  2.4. Quantitative histological analysis
  Mice were anaesthetized and sacrificed after echocardiography analysis. The hearts were excised and weighed immediately after being washed with cold phosphate-buffered saline (PBS). The cardiac tissues for histological and immunohistochemistry analysis were fixed with 4% paraformaldehyde for 12 h, dehydrated in 20% sucrose for 24 h and then embedded in paraffin. Serial sections (5μm thick) were stained with hematoxylin and eosin (H & E) for morphological analysis and picrosirius red for the detection of fibrosis. For morphometrical analysis, photographs of left ventricular sections cut from the same location of each heart were observed under 4 times, 200 times, 400 times magnification, respectively (Leica Microsystems Imaging Solutions Ltd, Cambridge, UK). Myocyte cross sectional area was measured by Image Pro Plus using the photographs under 400 times. Interstitial fibrosis was visualized with picrosirius red staining, and the cardiac fibrosis volume fraction was calculated as the ratio of the stained fibrotic area to the total myocardial area.


  2.5. Western Blot analysis
  The cardiac tissues were cracked by Tissue homogenizer and Ultrasonic unit. After centrifugation (12000rpm, 15min, 4°C), the protein content of supernatant was determined by BCA protein quantitative method. The loading quantity of samples was 30μg. Samples were separated by 12% SDS-PAGE and transferred to PVDF membranes. After blocked, blots were probed with the appropriate primary antibodies overnight at 4 °C, then washed and incubated with horseradish peroxidase-conjugated secondary antibody. Bands were visualized by use of a super Western sensitivity chemiluminescence detection system (Pierce). The conditions of primary and secondary antibodies used for immunoblot analysis were summarized in table two.


  2.6. Quantitative Real-Time PCR
  Total RNA was isolated from heart tissues using Trizol Reagent (Invitrogen). The complementary DNA was synthesized using the Kit (017317, Promega, USA). Relative quantitation by real-time PCR was performed using SYBR Green to detect PCR products in real time with ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Primer sequences were as follows: EIF5:5′-CAGAAAAACAAGAGCGAAGACAG-3′,5′-GCTTCCAGAGACAACTCCTCC-3′;TNF-α:5′-CCCACGTCGTAGCAAACCA-3′,5′-ACAAGGTACAACCCATCGGC-3′;IL-1β:5′-TGCCACCTTTTGACAGTGATG-3′,5′-AAGGTCCACGGGAAAGACAC-3′;IL-6:5′-CGGCCTTCCCTACTTCACAA-3′,5′-TTCTGCAAGTGCATCATCGT-3′;β1-AR:5′-GCCCTTTCGCTACCAGAGTT-3′,5′-ACTTGGGGTCGTTGTAGCAG-3′;β2-AR:5′-TCGAGCGACTACAAACCGTC-3′,5′-AAGTCCAGAACTCGCACCAG-3′,ANF:5′-CTTCCAGGCCATATTGGAG-3′,5′-GGGGGCATGACCTCATCTT-3′;BNP:5′-ACAAGATAGACCGGATCGGA-3′,5′-AGCCAGGAGGTCTTCCTACA-3′;CollagenI:5’-GTAACTTCGTGCCTAGCAACA-3’,5’-CCTTTGTCAGAATACTGAGCAGC-3’;CollagenIII:5’-CCTGGCTCAAATGGCTCAC-3’;5’-CAGGACTGCCGTTATTCCCG-3’.PCR program was performed under the following conditions: 95 °C for 2 min, followed by 40 amplification cycles (95°C for 15 s, 60 °C for 1min ). The CT (threshold cycle) values obtained for genes of interest were normalized to concurrent measurement of EIF5 mRNA level, and fold changes were compared to the control.


  2.7. ICP-MS
  The concentrations of AuNPs in tissues were assessed by quantitative inductively coupled plasma mass spectrometry (ICP-MS). Cardiac tissues (approximately 30 mg each) were digested in aqua fortis (nitric acid:hydrochloric acid 3:1). After adjusting the solution volume to 2 ml using 2% nitric acid and 1% hydrochloride acid (1:1), Au content assays were performed using an ELAN DRC e ICP-MS instrument (PerkinElmer, Massachusetts, USA).


  2.8. Statistical analysis
  Data was summarized as means ± SEM. Differences in data between groups were compared using Prism 5 (GraphPad Software Incorporae, La Jolla, CA, USA) with one-way ANOVA followed by Dunnett’s test. Data with P < 0.05 was considered statistically significant.

 

3.Results


  3.1.The accumulation of AuNPs in the mouse heart
  To investigate the effect of AuNPs on β-AR mediated cardiac remodeling, experiments were designed as the working flow chart shown in Fig. 1A. Firstly, the accumulation of AuNPs was determined by ICP-MS in mouse heart. As shown in Fig. 1B, accumulation of AuNPs in the heart was in a size-dependent manner. The 13 nm AuNPs showed the highest accumulation in heart (1438+236.9ng/g) which was extremely high in comparison with that of 30nm (68.76+17.33ng/g) and 50nm (18.21+4.052ng/g) .


  3.2.Effects of the AuNPs on cardiac hypertrophy
  The ratio of heart weight to tibia length (HW/TL), the ratio of heart weight to body weight (HW/BW), LVPW;d, myocyte cross sectional area, ANF, BNP are all the major indicators of cardiac hypertrophy. As shown in Fig. 2A and 2B, ISO could markedly increase LVPW;d and AuNPs (13, 30 and 50nm) could reverse this process. Similarly, AuNPs (13, 30 and 50nm) could attenuate HW/ BW (Fig. 2C) and HW/ TL (Fig. 2D) increased significantly by ISO. Moreover, AuNPs (13, 30 and 50nm) could reduce myocyte cross sectional area (Fig. 2E,F) increased by ISO. AuNPs (13, 30 and 50nm) could also decreased the mRNA expression of ANF(Fig. 2G) and BNP (Fig. 2H).


  3.3.Effects of the AuNPs on cardiac fibrosis
  Chronic long term ISO stimulation could cause fibrosis [21]. Our results indicated that acute short term ISO stimulation did not induce obvious fibrosis. Furthermore, AuNPs did not affect cardiac fibrosis , either (Fig. 3A, B). Collagen I and CollagenIII are also fibrosis markers. The mRNA expressions of Collagen I (Fig. 3C) and CollagenIII (Fig. 3D) consistent with the above conclusion.


  3.4. Effects of the AuNPs on cardiac functions
  Assessment of cardiac function is important in cardiac diseases. The left ventricular ejection fraction (EF) and fractional shortening (FS) were the most important indicators to evaluate the cardiac contraction function. Results showed that the EF and FS were still normal after exposure to ISO. AuNPs (13, 30 and 50nm) did not affect EF or FS (Fig. 4A , B). E/A, E’/A’ and E/E’ were used to evaluate cardiac diastolic function. Similar to cardiac contraction function, ISO did not affect cardiac diastolic function obviously in this model. AuNPs (13, 30 and 50nm) did not affect cardiac diastolic function, either (Fig. 4C-F).


  3.5. Effects of the AuNPs on β-AR mediated IL-6 mRNA expression
  First, H & E staining showed that ISO induced inflammatory cell infiltration. However, the additional accumulation of AuNPs in the heart did not aggravate ISO-induced cardiac inflammation (Fig. 5A). Furthermore, inflammatory cytokines TNF-α、IL-1β and IL-6, were detected with Real Time PCR. Consistent with cardiac hypertrophy, AuNPs (13, 30 and 50nm) inhibited IL-6 production increased by ISO significantly (Fig. 5B). In contrast to IL-6, ISO did not increase the production of TNF-α (Fig. 5C) and IL-1β (Fig. 5D).


  3.6. Effects of the AuNPs on β-AR mRNA expression
  It has been reported that chronic ISO stimulation downregulated the expression of both β1-AR and β2-AR [22]. In this study, the result showed that acute ISO stimulation upregulated β1-AR but not β2-AR mRNA expression (Fig.6A, B). AuNPs (13, 30 and 50nm) inhibited the upregulation of β1-AR mRNA expression significantly (Fig. 6A) but had no influence on β2-AR mRNA expression (Fig. 6B).


  3.7. Effects of the AuNPs on β-AR mediated ERK1/2 pathway activation
  It is well known that ERK1/2 MAPK pathway plays an important role in β-AR- mediated cardiac hypertrophy [23,24]. The results indicated AuNPs (13, 30 and 50nm) decreased the phosphorylation of ERK1/2 induced by ISO obviously (Fig. 7A, B) which consistent with the function of AuNPs in inhibiting β-AR-mediated cardiac hypertrophy.

 

Discussion


  In this study, we investigated the safety of AuNPs in the acute cardiac hypertrophic model induced by large dose of ISO. The result suggested that AuNPs could inhibit cardiac hypertrophy in this model. The mechanism may be related to the inhibition of β1-AR expression and its downstream signaling pathway such as inflammation and the phosphorylation of ERK1/2.

 

  Our study indicated that AuNPs (13, 30 and 50nm) inhibted the increase of β1-AR mRNA expression induced by ISO but not β2-AR, which is consistent with the result that β-AR-mediated cardiac hypertrophy is primarily due to the activation of β1-AR [25, 26]. Recent studies have proved that AuNPs could affect the synthesis and function of biological macromolecules. For example, AuNPs could interfer the synthesis of ribosomal protein [27]. Further, AuNPs could conjugated to G-protein and affect the activity of G-protein [28]. These studies suggested AuNPs may regulate the expression of β1-AR and its downstream signaling pathway which reflects the activity of β1-AR.

 

  Many studies suggested that inflammation is involved in cardiac hypertrophy. Pro-inflammatory cytokines such as IL-6, IL-1β and TNF-α promote cardiac hypertrophy [29, 30]. In this study, the mRNA expression of IL-1β and TNF-α did not increase but the mRNA expression of IL-6 increased after acute ISO treatment.  Some studies have indicated ISO could increase the expression of IL-6, IL-1β and TNF-α [31, 32]. However, it has also been reported that ISO treatment downregulated TNF-α production and caused no change in IL-6 production [33]. It suggested that inflammation is a complicated process and different inflammatory cytokines may have different reactions after β-receptor activation. In addition, the dose of ISO and the time course of administration may also have influence on the inflammatory cytokines’ production. The specific mechanisms still need more research. It has also been reported that AuNPs of 21nm could reduce IL-6 mRNA level in the fat [34]. Consistent with the previous study, in our study, AuNPs (13, 30 and 50nm) inhibited ISO-increased IL-6 mRNA expression which contributed to the β-AR-mediated cardiac hypertrophy. 

 

  ERK1/2 signaling pathways are important regulators of β1-AR-mediated cardiac hypertrophy [35-37]. In the present study, we found AuNPs inhibited the phosphorylation of ERK1/2 induced by ISO. In addition, recent work has suggested that ERK1/2 signaling is regulated, at least in part, by oxidative stress [38, 39] and that antioxidants can function to block the activation of ERK1/2 both in vitro and in vivo [40, 41]. While some other studies have shown that AuNPs could suppress oxidative stress and elevate the antioxidant defense system in vivo [42-45]. So AuNPs may inhibit the phosphorylation of ERK1/2 partly through the inhibition of oxidative stress.

 

  AuNPs(13,30,50nm) inhibited ISO-induced cardiac hypertrophy in the present study, but AuNPs have no effect on either the systolic or diastolic functions. It is noteworthy that both the systolic and diastolic functions did not change after ISO treatment. The possible reason is that cardiac remodeling is in a compensation stage, so both the systolic and diastolic functions are not damaged in this stage.

 

  There are some differences between the results of all the studies about the toxicity of AuNPs. It is likely due to their modifications, functional attachment of their surfaces, shapes and sizes [46, 47]. For example, known as a modifying polymer, polyethylene glycol (PEG) decreases immunogenicity and increases stability of drugs in the circulatory system. Therefore, the coated PEG reduces the chance of heart toxicity induced by gold nanoparticles [48-50].

 

  Taken together, our results showed that AuNPs inhibited cardiac hypertrophy mediated by β-AR and this effect depends on a complex mechanism involving inhibition of β1-AR expression and its downstream effectors IL-6 and ERK 1/2 (Fig. 8). These results raise the hope that AuNPs might be used as multiple-function materials (drug carrier systems and anti-cardiac hypertrophy agents) for cardiac diseases treatment.

 

Acknowledgments


  This work was supported by grants from the National Basic Research Program of China (Grant no. 2014CBA02000), the National Natural Science Foundation of China (81471893, 81270157, 91539123, 81070078) and Beijing Municipal Natural Science Foundation (7172235).

 

Conflicts of interest
None.

 

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Figure Legend


  Fig. 1. Experimental flow chart and Au accumulation in heart.
  (A) The experimental flow chart of the present study. (B) The Au content in mouse heart was determined with inductively coupled plasma mass spectrometry (ICP-MS). ***P < 0.001. Data represent means ± SEM.


Fig. 2. The effects of AuNPs on cardiac hypertrophy.
  (A) Representative M-mode echocardiography images were performed to show left ventricular wall thickness. (B) Quantitative analysis of diastolic left ventricular posterior wall thickness (LVPW;d). (C) Quantitative analysis of HW/BW ratio. (D) Quantitative analysis of HW/TL ratio. *P < 0.05, **P < 0.01, ***P < 0.001. Data represent means ± SEM. (E) Representative micrographs of myocyte cross sectional area. The scale bar is 50μm. (F) Quantitative analysis of myocyte cross sectional area. *P < 0.05, **P < 0.01, ***P < 0.001. (G) Quantitative analysis of ANF mRNA expression. *P < 0.05, ***P < 0.001. (H) Quantitative analysis of BNP mRNA expression. *P < 0.05.


Fig.3. The effects of AuNPs on cardiac fibrosis
  (A) Representative micrographs of picrosirius red-stained sections of the ventricle. Red parts represent collagen. The scale bars are 2 mm and 200μm. (B) Quantification of cardiac interstitial collagen content from picrosirius red-stained sections with results expressed as the ratio of collagen area to heart area. There is no significance among all the groups. (C) The mRNA expression of Collagen I in the heart tissue.  (D) The mRNA expression of CollagenIII in the heart tissue.


Fig. 4. The effects of AuNPs on cardiac function.
  (A) Left ventricular ejection fraction (EF) and (B) fractional shortening (FS) were measured toreflect cardiac contraction function. (C) Representative doppler echocardiographyic images. (D)E/A, (E) E’/A’ (F) and E/E’ ratios were used toreflect cardiac diastolic function.  None significances were found.


  Fig. 5. The effects of AuNPs on inflammation in heart.
  (A) Representative images showing hematoxylin and eosin (HE) staining of heart sections. The arrows refers to the infiltrated inflammatory cells. The scale bars of 12.5× images are 2 mm, of 250× images are 200μm and those of 500× images are 50μm. (B) The mRNA expression of IL-6 in the heart tissue. (C) The mRNA expression of TNF-α in the heart tissue. (D) The mRNA expression of IL-1β in the heart tissue. **P < 0.01, n.s. no significance. Data represent means ± SEM.


  Fig. 6. The effects of AuNPs on β-AR mRNA expression in heart.
  (A) The mRNA expression of β1-AR in the heart tissue. (B) The mRNA expression of β2-AR in the heart tissue. *P < 0.05,**P < 0.01, ns no significance. Data represent means ± SEM.


Fig.7. The effects of AuNPs on the phosphorylation of ERK1/2.
  (A) The protein expression of P-ERK1/2 (Phosphorylated ERK 1/2) and T-ERK1/2 (Totel ERK1/2 ) of all the groups. (B) Quantitative analysis of the level of P-ERK1/2 of all the groups. *P <0.05, **P < 0.01.


  Fig.8. Working model of the effects of AuNPs on β-AR mediated cardiac hypertrophy


Table 1. Treatment of animals of each group



Table 2. Conditions of first and second antibodies for western blot

 

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作者简介
李子健
单位:北京大学第三医院
简介:  心血管受体研究北京市重点实验室副主任,   中国病理生理学会受体信号转导专业委员会副主任委员,
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