Abstract
Atherosclerosis, a multifactorial chronic inflammatory response, is closely associated with oxidatively modified low-density lipoprotein (ox-LDL). High-mobility group box 1 (HMGB1) is a DNA-binding protein, which upon release from cells exhibits potent inflammatory action. Insulin-like growth factor 1 (IGF-1) can elicit a repertoire of cellular responses including proliferation and anti-apoptosis. However, the role of IGF-1 in inflammation is still unclear. In the present study, we aimed to investigate the role of IGF-1 in inflammation and the underlying mechanism. Human aortic endothelial cells were stimulated by ox-LDL (50 μg/ml) to induce inflammation. The expression of intercellular adhesion molecule 1 (ICAM-1) was assessed by western blot analysis and immunofluorescence. The release of HMGB1 was determined by enzyme-linked immunosorbent assay. IGF-1 receptor (IGF-1R) expression was assessed by reverse transcription-polymerase chain reaction and western blot analysis. IGF-1R phosphorylation was determined by western blot analysis. Ox-LDL stimulation reduced IGF-1R mRNA and protein expression but increased HMGB1 release. IGF-1 treatment decreased ox-LDL-induced ICAM-1 expression potentially through reducing HMGB1 release, while picropodophyllin, an IGF-1R specific inhibitor, increased the inflammatory response. In conclusion, IGF-1 can alleviate ox-LDL-induced inflammation by reducing HMGB1 release, suggesting an unexpected beneficial role of IGF-1 in inflammatory disease.
Introduction
Atherosclerosis is regarded as a multifactorial disease. Chronic inflammation plays a critical role in every stage of atherosclerosis, from initiation to progression, and eventually plaque rupture [1,2]. Oxidatively modified low-density lipoprotein (ox-LDL) is a hypothetical risk factor for atherosclerotic inflammation [2]. Inflammatory stimulation provokes changes in the phenotype of the cells of the arterial wall that allow penetration of inflammatory cells and LDL particles across the endothelial barrier, trapping them in the subendothelial space [3,4]. An inflammatory reaction then occurs in the subendothelial space and leads to generation of reactive oxygen and nitrogen species. In this setting, LDL particles become modified to a form known as ox-LDL [5].
High-mobility group box 1 (HMGB1) is a non-histone nuclear protein implicated as a mediator of tissue damage and inflammation [6]. It was originally identified as a multifunctional transcription factor by binding to their cognate DNA sequences [7]. The translocation of HMGB1 from the inside to the outside of the cell plays an important role in host defense and inflammation and usually occurs in two different ways [8]. First, HMGB1 acts as a cytokine which can be actively secreted by inflammatory cells after stimulation with exogenous pathogen-derived molecules or endogenous inflammatory mediators [9–12]. Secondly, it is also passively released by necrotic cells, triggering inflammatory responses through binding the receptor for advanced glycation endproduct (RAGE) and activating nuclear factor kappa B (NF-κB) [13–15]. HMGB1 elicits pro-inflammatory responses in endothelial cells [16] and induces the migration and proliferation of vascular smooth muscle cells [17]. It has been implicated in a number of inflammatory diseases such as septic shock, acute lung inflammation, and atherosclerosis [18–21].
Insulin-like growth factor-1 (IGF-1), a peptide hormone, is synthesized in liver and kidney and has pleiotropic effect on cell growth and metabolism [22]. The intracellular signal pathways involved in IGF-1 transduction consist of insulin receptor substrate-1, phosphatidylinositol (PI) 3-kinase, Akt, and mitogen-activated protein kinase cascade [23]. It has been demonstrated that constitutive overexpression of IGF-1 can protect myocytes from death and limit ventricular dilation and cardiac hypertrophy in the viable myocardium after infarction [24]. The role of IGF-1 in the cell survival has been demonstrated, but its effect on the inflammation has not been fully investigated.
In the present study, we aimed to investigate the relationship among ox-LDL, HMGB1, and IGF-1 during inflammation in human aortic endothelial cells (HAECs). We demonstrated that ox-LDL can increase HMGB1 release and decrease IGF-1R expression. IGF-1 can decrease ox-LDL-induced ICAM-1 expression by reducing HMGB1 release.
Materials and Methods
Cell culture and treatment
HAECs purchased from the American Type Cell Collection (Manassas, USA) were cultured in endothelial cell medium (ScienCell, Carlsbad, USA) containing 5% fetal bovine serum (Gibco, Carlsbad, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37°C. Cells were cultured up to the fourth passage for experiments. HAECs were stimulated by ox-LDL (50 μg/ml; Yiyuan Biotechology, Guangzhou, China) to induce inflammation. To analyze whether IGF-1 alleviates ox-LDL-induced inflammation, cells were pretreated with or without picropodophyllin (PPP) (1 μM; Santa Cruz Biotechnology, Paso Robles, USA), and then with IGF-1 (100 ng/ml) for 1 h. To inhibit HMGB1 expression, HAECs were transiently transfected with negative control of siRNA or HMGB1 siRNA (10 ng; GenePharma, Shanghai, China) in Optimem Medium (Invitrogen, Carlsbad, USA) by using Lipofectamine™ 2000 (Invitrogen). The following siRNA sequences for HMGB1 gene were used: forward, 5′-AAUAGGAAA AGGAUAUUGCU-3′; reverse, 5′-AGCAAUAUCCUUU UCCUAUU-3′.
Reverse transcription-PCR
Total RNA was extracted with Trizol reagent (Invitrogen) from HAECs. Purified RNA (1 μg) was treated with DNase and reverse transcribed (RevertAid M-MulV Reverse Transcriptase; Fermentas, St Leon-Rot, Germany) following the manufacturer's protocol. Polymerase chain reaction (PCR) was performed with the 7500 Real-Time PCR System (Applied Biosystems, Hercules, USA). Three technical replicates were run for each gene in each sample. The primers for intercellular adhesion molecule 1 (ICAM-1) were as follows: forward, 5′-TTGGAAGCC TCATCCG-3′; reverse, 5′-CAATGTTGCGAGACCC-3′; for IGF-1R: forward, 5′-AAAGAATTCAGTGTGTGG CGGCGGCGG-3′; reverse, 5′-AAAGTCGACTCCTTTTA TTTGGGACGA-3′; and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH): forward, 5′-AGGTCGGTGTGAACGGATTTG-3′; reverse, 5′-TGTAGACCATGTAGTTGAGGTCA-3′. PCR amplification was at 95°C for 5 min, 35 cycles at 95°C for 10 s, annealing at 56°C for 30 s and elongation at 72°C for 30 s. The mRNA expression of ICAM-1 and IGF-1R was normalized to that of GAPDH.
Western blot analysis
Protein was extracted from cells with the RIPA lysis buffer (Beyotime, Nantong, China) and assayed by the BCA protein assay kit (Beyotime). Equal amounts of protein (2 mg/ml) were separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Millipore, Billerica, USA). After being blocked with 5% non-fat milk, the blots were washed in Tris-buffered saline with Tween (TBS-T) three times (10 min each) and then incubated at 4°C overnight with appropriate primary antibodies: rabbit anti-β-actin (1 : 1000; Cell Signaling Technology, Danvers, USA), rabbit anti-IGF-1R (IGF-1R) or rabbit anti-p-IGF-1R (both 1 : 1000; Cell Signaling Technology), rat anti-HMGB1 (1 : 500; Abcam, Cambridge, USA), or mouse monoclonal anti-ICAM-1 (1 : 500; Santa Cruz Biotechnology). The blots were then washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (Jingmei Biotech, Shanghai, China) for 2 h at room temperature. After three washes in TBS-T, the membrane was visualized by enhanced chemiluminescence plus reagents (Millipore).
Immunocytochemistry
HAECs were fixed in 4% paraformaldehyde and permeabilized in phosphate-buffered saline containing 0.5% Triton X-100. After being blocked with normal serum, cells were incubated with goat anti-ICAM-1 (1 : 200; Santa Cruz Biotechnology) overnight at 4°C. Alexa 555-conjugated donkey anti-goat IgG (1 : 2,000; Invitrogen) was used as secondary antibody. A drop of Prolong Gold Antifade reagent with 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen) was used to seal coverslips. Images were acquired by laser scanning confocal microscopy (LSM710; Carl Zeiss, Ostalbkreis, Germany) and analyzed with Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, USA).
Enzyme-linked immunosorbent assay
The supernatant was obtained by centrifugation at 100 g for 10 min. The HMGB1 levels in supernatant were determined by using the HMGB1 ELISA kit (Uscn Life Science Inc., Wuhan, China) according to the manufacturer's instructions. All operations were performed at room temperature. Mean absorbance for standards and samples was determined in duplicate. The color reaction was detected by using Varioskan Flash multifunction plate reader (Thermo Scientific, Rockford, USA).
Statistical analysis
Data are expressed as mean ± SD. SPSS 16.0 for Windows (SPSS, Chicago, USA) was used for statistical analysis. Differences for two groups were compared by Student's t-test and for more than two groups by analysis of variance. P< 0.05 was considered statistically significant.
Results
ICAM-1 expression was increased after ox-LDL stimulation
To assess the expression of ICAM-1, HAECs were stimulated by ox-LDL (50 μg/ml) for indicated time periods and then total protein and RNA were extracted. The western blot analysis showed that the ICAM-1 expression was significantly increased after 4 h stimulation, with a peak at 12 h stimulation [Fig. 1(A,B)]. The reverse transcription (RT)-PCR results showed the similar results as well [Fig. 1(C)]. Therefore, HAECs stimulated by 50 μg/ml ox-LDL for 12 h were used in the following experiment.
ICAM-1 mRNA and protein expression was induced by ox-LDL HAECs were stimulated by 50 μg/ml ox-LDL for different time points and ICAM-1 mRNA and protein expression was detected. (A,B) Western blot analysis of ICAM-1 protein expression. (C) Quantification of RT-PCR results of ICAM-1 mRNA expression. Values are expressed as mean ± SD from three independent experiments. *P< 0.05 vs. 0 h treatment.
Ox-LDL stimulation increased HMGB1 release
The translocation of HMGB1 from the inside to the outside of cells plays an important role in inflammatory response. We investigated the role of HMGB1 in ox-LDL-induced ICAM-1 protein expression. HAECs were transiently transfected with HMGB1 siRNA and then stimulated with ox-LDL. Compared with the control, ICAM-1 expression was increased by 12 h ox-LDL treatment, while it was decreased by pretreatment with HMGB1 siRNA [Fig. 2(A,B)]. Then we investigated the HMGB1 release after ox-LDL stimulation by enzyme-linked immunosorbent assay (ELISA). The results showed that HMGB1 release was rarely detected in control cells, but ox-LDL could significantly increase the HMGB1 release [Fig. 2(C)]. HMGB1 siRNA pretreatment decreased the release of HMGB1 induced by ox-LDL.
Ox-LDL-induced ICAM-1 protein expression and HMGB1 release HAECs were transfected with HMGB1 siRNA and then stimulated by 50 μg/ml ox-LDL for 12 h. ICAM-1 protein expression and HMGB1 release were detected by western blot analysis and ELISA, respectively. (A,B) Western blot analysis of ICAM-1 protein expression. (C) ELISA analysis of HMGB1 release. Values are expressed as mean ± SD from three independent experiments. *P< 0.05 vs. control; #P< 0.05 vs. ox-LDL. NC, negative control of HMGB1 siRNA.
Ox-LDL stimulation decreased IGF-1R mRNA and protein expression
IGF-1R mRNA and protein expression was determined in HAECs after 50 μg/ml ox-LDL stimulation for 12 h. IGF-1R mRNA expression was significantly decreased by ox-LDL [Fig. 3(A)]. Similarly, ox-LDL markedly reduced IGF-1R protein expression when compared with control cells [Fig. 3(B,C)]. As a transmembrane tyrosine kinase, membrane-bound β-subunit of IGF-1R becomes autophosphorylated and activates subsequent signaling pathway when ligand interacts with the IGF-1R α-subunit. Therefore, we detected IGF-1R phosphorylation by western blotting. The results showed that ox-LDL had no significant effect on IGF-1R phosphorylation [Fig. 3(B,D)].
Ox-LDL-induced IGF-1R mRNA and protein expression as well as its phosphorylation HAECs were stimulated by 50 μg/ml ox-LDL for 12 h and IGF-1R mRNA and protein expression and its phosphorylation was detected. (A) Quantification of RT-PCR results of IGF-1R mRNA expression induced by ox-LDL. (B–D) Western blot analysis of IGF-1R and phosphorylated IGF-1R (p-IGF-1R) protein expression induced by ox-LDL. Values are expressed as mean ± SD from three independent experiments. *P< 0.05 vs. control.
IGF-1 increased phosphorylation of IGF-1R
We then investigated the effect of IGF-1 and PPP on the expression of IGF-1R after 50 μg/ml ox-LDL stimulation for 12 h. Compared with the ox-LDL group, IGF-1 significantly increased the phosphorylation of IGF-1R [Fig. 4(A,B)]. However, PPP, an IGF-1R inhibitor, reduced the IGF-1-induced IGF-1R phosphorylation. IGF-1 and PPP had no effect on the IGF-1R expression [Fig. 4(A,C)].
IGF-1 increased phosphorylation of IGF-1R HAECs were stimulated with or without PPP (1 μM) before addition of IGF-1 (100 ng/ml). After 1 h incubation, ox-LDL (50 μg/ml) was added for 12 h. IGF-1R and phosphorylated IGF-1R (p-IGF-1R) protein expression was detected by western blot analysis. Values are expressed as mean ± SD from three independent experiments. *P< 0.05 vs. ox-LDL; #P< 0.05 vs. ox-LDL+IGF-1. PPP, picropodophyllin.
IGF-1 attenuated ox-LDL-induced inflammation and HMGB1 release
To investigate whether IGF-1 attenuated ox-LDL-induced inflammation, HAECs were first treated with IGF-1 for 1 h and then with ox-LDL for 12 h. The results showed ICAM-1 expression was decreased by IGF-1 compared with the ox-LDL group [Fig. 5(A,B)]. Pretreatment with PPP before addition of IGF-1 significantly elevated ICAM-1 expression. The above results suggested that IGF-1 could alleviate the inflammatory response induced by ox-LDL.
ICAM-1 protein expression and HMGB1 release were reduced by IGF-1 after ox-LDL stimulation HAECs were stimulated with or without PPP (1 μM) before addition of IGF-1 (100 ng/ml). After 1 h incubation, ox-LDL (50 μg/ml) was added for 12 h. ICAM-1 protein expression and HMGB1 release were detected by immunofluorescence and ELISA, respectively. (A,B) Immunofluorescence of ICAM-1 protein expression. Nuclei were labeled with DAPI (blue); ICAM-1 was stained with goat anti-ICAM-1 primary antibody and Alexa 555-conjugated donkey anti-goat second antibody (red). (C) Quantification of HMGB1 release by ELISA. Values are expressed as mean ± SD. *P< 0.05 vs. ox-LDL; #P< 0.05 vs. ox-LDL+IGF-1. PPP, picropodophyllin.
We then investigated whether HMGB1 was involved in the anti-inflammation of IGF-1 by ELISA. The results showed HMGB1 release induced by ox-LDL was significantly reduced after IGF-1 treatment [Fig. 5(C)], but it was increased by pretreatment with the PPP. It suggested that IGF-1 decreased ox-LDL-induced inflammation by reducing the release of HMGB1 in HAECs.
Discussion
As we all know, atherosclerosis is a chronic inflammatory disease that is often associated with multiple risk factors, such as dyslipidemia. Without effective treatment, atherosclerosis can result in cardiovascular disease, which is still the primary cause of morbidity and mortality in the world [25]. ox-LDL are the major contributor to vascular inflammation during atherosclerosis. Ox-LDL and other lipid particles stimulate the inflammatory cells to secrete various inflammatory cytokines, which lead to chemiotaxis and migration of more inflammatory cells and a vicious cycle. In the present study, we found that ox-LDL could increase ICAM-1 expression in a time-dependent manner. Moreover, HMGB1 played a critical role in ox-LDL-induced inflammation. Ox-LDL stimulation increased HMGB1 release from the cells to supernatant, and inhibition of HMGB1 by siRNA can reduce ICAM-1 expression induced by ox-LDL.
HMGB1, a non-histone chromatin associated nuclear protein, was discovered as a specific regulator of gene expression [26]. It is constitutively expressed in quiescent cells stored in the nucleus [27]. As a nuclear protein, HMGB1 is implicated in various cellular functions, including the regulation of nucleosomal structure and stability, and transcription factors [28]. HMGB1 has three major protein domains consisting of two DNA binding domains (A- and B-box) and the C-terminal which is important for the transcription stimulatory function [29,30]. The B box domain plays an important role in the pro-inflammatory activity of HMGB1 [31]. There are two distinct mechanisms for cell to release HMGB1. The first mechanism is that necrotic cells can passively release HMGB1 into extracellular milieu. The second mechanism is the active secretion of HMGB1 by activated immune cells [32]. Released HMGB1 exerts paracrine and autocrine activity and enhances pro-inflammatory and innate immune response to initiate adaptive immune response [33]. Increased expression of HMGB1 was detected in human atherosclerotic lesions [20].
Although ox-LDL and HMGB1 play an important role in atherosclerosis, few studies have been done on the relationship between ox-LDL and HMGB1. In our study, we found that ox-LDL could increase the release of HMGB1. Recent studies show that the lectin-like oxidized LDL receptor-1 facilitates the uptake of ox-LDL and mediate ox-LDL-induced expression of adhesion molecules via activation of NF-κB [34]. The release of HMGB1 induced by ox-LDL may be another mechanism of pro-inflammatory effect of ox-LDL.
The IGF-1 is essential for normal fetal and postnatal growth and development. Targeted deletion of IGF-1 results in growth deficiency, death shortly after birth, and impaired postnatal growth and infertility in mice [35,36]. The IGF-1 receptor (IGF-1R), a member of a family of transmembrane tyrosine kinases, binds IGF-1 with high affinity and initiates the physiological response to this ligand [37]. Phosphorylation of IGF-1R can activate the PI3-kinase/Akt pathway which plays an important role in cell survival. Overexpression of IGF-1 abolishes the necrosis and apoptosis of myocytes [24]. Cardiac overexpression of IGF-1R protects against left ventricular diastolic dysfunction and remodeling induced by diabetes [38]. However, the role of IGF-1 in inflammation response is still unclear. In our experiments, ox-LDL stimulation decreased IGF-1R mRNA and protein expression, with an increased HMGB1 release and ICAM-1 expression in HAECs. Considering the pro-inflammatory activity of HMGB1, we speculated that IGF-1 might play an anti-inflammatory role in ox-LDL-induced inflammation by decreasing HMGB1 release. We then used PPP, a specific IGF-1R inhibitor, to verify this hypothesis. IGF-1 treatment increased IGF-1R phosphorylation and decreased HMGB1 release and ICMA-1 expression, while inhibition of IGF-1R by PPP aggravated the inflammation response. The RAGE, a multiligand cell surface molecule, is a main receptor for HMGB1. The binding of HMGB1 to RAGE leads to the activation of NF-κB and inflammatory cytokine expression [39]. Our results showed IGF-1 decreased the release of HMGB1 and expression of ICAM-1, which meant a reduced number of HMGB1 that could bind to RAGE, which may be the mechanism of anti-inflammatory effect of IGF-1. The mechanism of decreased release of HMGB1 by IGF-1 needs further investigation.
In conclusion, we found that ox-LDL could decrease IGF-1R mRNA and protein expression and induce ICAM-1 expression by increasing HMGB1 release. IGF-1 treatment could increase IGF-1R phosphorylation and reduce HMGB1 release to alleviate ox-LDL-induced inflammation. IGF-1 will be an effective target for the treatment of inflammation and atherosclerosis.
Funding
This work was supported by the grants from the National Natural Science Foundation of China (30571844) and Shandong Province Natural Science Foundation of China (2009ZRB14005).
Acknowledgement
We would like to thank Xiao Wu from Shandong University for technical assistance.
