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. 2015 Feb 27;593(Pt 8):1873–1886. doi: 10.1113/jphysiol.2014.287045

Actin cytoskeletal rearrangement and dysfunction due to activation of the receptor for advanced glycation end products is inhibited by thymosin beta 4

Sokho Kim 1, Jungkee Kwon 1,✉
PMCID: PMC4405748  PMID: 25640761

Abstract

The receptor of advanced glycation end products (RAGE) is a cell-surface receptor that is a key factor in the pathogenesis of diabetic complications, including vascular disorders. Dysfunction of the actin cytoskeleton contributes to disruption of cell membrane repair in response to various type of endothelial cell damage. However, mechanism underlying RAGE remodelling of the actin cytoskeleton, by which globular actin (G-actin) forms to filamentous actin (F-actin), remains unclear. In this study we examined the role of thymosin beta 4 (Tβ4) – which binds to actin, blocks actin polymerization, and maintains the dynamic equilibrium between G-actin and F-actin in human umbilical vein endothelial cells (HUVECs) – in the response to RAGE. Tβ4 increased cell viability and decreased levels of reactive oxygen species in HUVECs incubated with AGEs. Tβ4 reduced the expression of RAGE, consistent with a down-regulation of the F-actin to G-actin ratio. The effect of remodelling of the actin cytoskeleton on RAGE expression was clarified by adding Phalloidin, which stabilizes F-actin. Moreover, small interfering RNA was used to determine whether intrinsic Tβ4 regulates RAGE expression in the actin cytoskeleton. The absence of intrinsic Tβ4 in HUVECs evoked actin cytoskeleton disorder and increased RAGE expression. These findings suggest that regulation of the actin cytoskeleton by Tβ4 plays a pivotal role in the RAGE response to AGEs.

Key points

  • Thymosin beta 4 (Tβ4) attenuates the vascular cellular toxicity induced by advanced glycation end products (AGEs) in human umbilical vein endothelial cells (HUVECs).

  • Tβ4 reduces expression of both the receptor of AGEs (RAGE) and the filamentous actin (F-actin) to globular actin (G-actin) ratio.

  • RAGE expression was regulated by actin cytoskeleton involved in Tβ4.

  • Tβ4 attenuates the vascular cellular toxicity induced by AGEs via remodelling of the actin cytoskeleton.

  • AGEs attenuate vascular-like tube formation of HUVECs, which is reversed by Tβ4 via remodelling of the actin cytoskeleton.

Introduction

Vascular disorders are major causes of mortality in diabetes with ageing (Frohlich-Reiterer & Borkenstein, 2010; Jenkins et al. 2010). Retinopathy, nephropathy and neuropathy are associated with pathological degeneration in vascular disorders (van den Oever et al. 2010; Wong et al. 2010), and many studies have demonstrated that dysfunction of vascular endothelial cells induced by advanced glycation end products (AGEs) plays a crucial role in the pathogenesis of diabetic complications (Candido et al. 2003; Bidasee et al. 2004). AGEs are a heterogeneous group of complex compounds that are synthesized from proteins or lipids that are formed irreversibly in diabetic conditions via a chain of non-enzymatic chemical reactions (Vlassara et al. 2008). They accumulate rapidly in the serum and in various tissues (Brownlee et al. 1988), mainly in the vascular walls, indicating that they affect the structure and functions of vascular endothelial cells via interactions with the cell-surface receptor for AGEs (RAGE) (Brett et al. 1993; Sengoelge et al. 1998).

The activation of RAGE promotes apoptosis, which results in various pathological effects and consequent diabetic complications (Busch et al. 2010). Previous studies have shown that the mechanism underlying the observed endothelial cell dysfunction in diabetic complications is RAGE-induced apoptosis (Bento et al. 2010), and that proinflammatory cytokines and reactive oxygen species (ROS) are generated during this process (Roszer, 2011). The accumulation of AGEs increases endothelial permeability and induces a series of changes in endothelial cell morphology and function (Soro-Paavonen et al. 2010). It has been reported recently that the interaction between AGEs and RAGE disrupts tight junctions and adherens junctions in human umbilical vein endothelial cells (HUVECs) via increased endothelial permeability, which is related to disorganization of the actin cytoskeleton (Hirose et al. 2010). Moreover, overexpression of RAGE significantly disorganizes the filamentous actin (F-actin) stress fibres, resulting in inhibition of plasma membrane resealing (Xiong et al. 2011). Therefore, prevention of RAGE activation is a major goal of treatments for diabetic vascular disorders. However, the mechanism between RAGE and remodelling of the actin cytoskeleton, which involves transformation of globular actin (G-actin) to F-actin, remains unclear.

Thymosin beta 4 (Tβ4) is a small (approximately 5 kDa) and highly conserved water-soluble peptide (Goldstein et al. 2012). It binds to actin, blocks actin polymerization and is the major actin sequestering molecule in mammalian cells (Sanger et al. 1995). Tβ4 regulates the dynamics of the actin cytoskeleton by maintaining a large pool of actin monomers that interact with G-actin to form a 1:1 complex for control of the assembly of F-actin (Safer et al. 1991). It is well established that cell differentiation, morphogenesis, migration and survival are affected by Tβ4, and that it maintains a dynamic equilibrium between G-actin and F-actin, which is fundamental for rapid reorganization of the cytoskeleton. Tβ4 is an angiogenic factor for early genes induced in endothelial cell differentiation in vitro (Grant et al. 1995), and is involved in the enhancement of vasculogenesis during development (Smart et al. 2007). Moreover, our previous findings indicate that the burn wound healing effect of Tβ4 in diabetic mice involves a reduction of RAGE (Kim & Kwon, 2014b). This prompted us to investigate the fundamental role of Tβ4 in the interplay between the actin cytoskeleton and RAGE.

The aim of the present study was to determine whether treatment with Tβ4 protects vascular endothelial cells against the interaction between RAGE and AGEs, and whether Tβ4 serves as a homeostasis mediator to regulate the levels of RAGE via remodelling of the actin cytoskeleton.

Methods

Chemicals

Tβ4 was purchased from Tocris Bioscience (Bristol, UK). Phalloidin, saline, BSA and other standard reagents were purchased from Sigma (St Louis, MO, USA). AGEs (modified with BSA) were purchased from BioVision (Milpitas, CA, USA). Primary antibodies raised against RAGE, protein kinase B (AKT), phosphorylated (p-)AKT, glycogen synthase kinase (GSK)3β, p-GSK3β, vascular endothelial growth factor receptor (VEGFR)2, p-VEGFR2 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies raised against VEGFR1 and p-VEGFR1 were purchased from Abcam (Cambridge, UK), the Tβ4 antibody was purchased from Millipore (Temecula, CA, USA), and the G-Actin/F-Actin In Vivo Assay Biochem Kit was purchased from Cytoskeleton (Denver, CO, USA). Secondary antibodies (i.e. anti-rabbit, anti-goat and anti-mouse IgG antibody conjugated with horseradish peroxidase) were obtained from Millipore. All other chemicals and reagents were of analytical grade.

Cell culture and treatments

HUVECs as primary cells (Lifeline Cell Technology, Frederick, MD, USA) were cultured at 37°C in a humidified atmosphere containing 5% CO2 in VascuLife complete medium (Lifeline Cell Technology). The HUVECs were incubated for 4 days until confluence, after which they were starved in serum-free basal medium for 24 h. Cells treated with BSA were regarded as control. Cells in the AGEs-treated groups were incubated with AGEs at various concentrations (100, 200, 300 and 500 μg ml−1) for different periods (24, 48 and 72 h), while cells in the experimental groups were pretreated with or without Tβ4 (0.01, 0.1, 0.3 and 0.5 μg ml−1) for 1 h before AGEs treatment. Cells in the Phalloidin-treated groups were incubated with Phalloidin at various concentrations (10, 50 and 100 μm) for 48 h, with or without Tβ4 for 1 h before AGEs treatment.

Small interfering RNA (siRNA) transfection

Cells were plated in 6 cm2 dishes (4 × 105 cells per dish) until confluence, and then starved for 24 h. The DharmaFECT 1 siRNA Transfection Reagent (Dharmacon, Denver, CO, USA) was used to transfect the cells with 50 nm Tβ4 siRNA, or scrambled siRNA oligonucleotides (Dharmacon) according to the manufacturer's instructions.

Cell viability assay

Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit from Sigma according to the manufacturer's instructions. Cells were grown on 96-well plates at a density of 2 × 104 cells per well. After treatment cell viability was evaluated by assaying for the ability of functional mitochondria to catalysse the reduction of MTT to a formazan salt by mitochondrial dehydrogenases. The index of cell viability was quantified using a multiplate reader spectrophotometer (PowerWave 2, Bio-Tek Instruments, Winooski, VT, USA) based on the absorbance at 570 nm.

Intracellular ROS assay

The level of ROS was quantified by fluorescence using 2′,7′-dichlordihydrofluorescin diacetate (DCF-DA; Invitrogen, Carlsbad, CA, USA). Cells were grown on 48-well plates (4 × 104 cells per well) and incubated under the corresponding treatment condition for 3 h. After the incubation period, cells were washed with PBS and stained with DCF-DA in PBS for 30 min in the dark. They were then washed twice with PBS and extracted with 0.1% Tween-20 in PBS for 10 min at 37°C. Fluorescence was recorded using an excitation wavelength of 490 nm and an emission wavelength of 525 nm.

Measurement of the F-actin/G-actin ratio

The amounts of intracellular G-actin and F-actin were measured using the G-Actin/F-Actin In Vivo Assay Biochem Kit (Cytoskeleton). Protein samples were obtained from the cells and tissues, and G-actin and F-actin were separated out by following the manufacturer's protocol. The total protein concentration in each tube containing either G-actin or F-actin was determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA), and the separated actin contents were analysed by immunoblotting. The F-actin to G-actin ratio was calculated by dividing the F-actin density regulated by the G-actin binding, and is expressed as a percentage of that in the control group.

Immunoblotting analysis

Proteins in the cells were subjected to SDS-PAGE using 8, 10 and 14% gels, and electrophoretically transferred to PVDF membranes (Bio-Rad). The membranes were blocked with 5% skimmed milk in PBS and then incubated individually with each primary antibody diluted to 1:1000 in 1% skimmed milk in PBS overnight at 4°C. The blots were further incubated with each secondary antibody diluted to 1:10 000 at room temperature for 1 h. The immunoreactions were visualized using the SuperSignal West Dura Extended Duration Substrate (Thermo Fischer Scientific, San Jose, CA, USA) and analysed using the ChemiImager system (Alpha Innotech, San Leandro, CA, USA).

Tube formation assay

The formation of vascular-like structures by HUVECs on growth factor reduced Matrigel (BD Biosciences, Bedford, MA, USA) was confirmed according to the manufacturer's protocol. Briefly, Matrigel was thawed at 4°C overnight, and each well of pre-chilled 96-well culture plates was coated with 50 μl Matrigel and incubated at 37°C for 1 h. HUVECs (5 × 103 cells) were added in 100 μl culture medium with various conditions. After 12 h of incubation at 37°C and 5% CO2 atmosphere, tube formation was observed and photographed using an inverted phase contrast microscope (Observer.A1, Carl Zeiss, Oberkochen, Germany). The degree of tube formation was quantified by measuring the length of tubes in three randomly chosen low power fields (100×) from each well using the Image Pro analysis program.

Statistical analyses

The data were analysed using Student's t test (for two groups), one-way ANOVA and Tukey's test (for more than two groups), and are presented as mean and SEM values. All samples were analysed in different experiments performed three times individually. The cutoff for statistical significance was set at P < 0.05. All analyses were performed using the Statistical Package for Social Sciences (version 13.0 for Windows, SPSS, Chicago, IL, USA).

Results

Tβ4 attenuates the vascular cellular toxicity induced by AGEs in HUVECs

The optimum concentration of AGEs was first established by examining the cell viability for AGEs present at various concentrations (100, 200, 300 and 500 μg ml−1) and for different incubation periods (24, 48 and 72 h; Fig.1A). Cell viability after 48 h was decreased for AGEs concentrations over 300 μg ml−1; a significant reduction of approximately 24% was revealed. Optimum stimulation was achieved using 300 μg ml−1 AGEs for 48 h, and so these parameters were used for further experiments. The cell viability of Tβ4-treated HUVECs with 300 μg ml−1 AGEs increased significantly in a dose-dependent manner; a significant increment of approximately 93% was revealed with 0.3 μg ml−1 Tβ4 (Fig.1B). The ROS assay was used as an indirect indicator of cell stress induced by AGEs. As shown in Fig.1C, AGEs-induced ROS were significantly attenuated by treatment with Tβ4 at concentrations >0.1 μg ml−1. Based on these results, the optimal concentration of Tβ4 required to attenuate AGEs-induced toxicity was 0.3 μg ml−1, and this was used in subsequent experiments. VEGFR1 and VEGFR2 in vascular endothelial cells are receptor proteins that induce vasculogenesis when combined with VEGF, an angiogenic growth factor. It has been reported recently that AGEs induce abnormal angiogenesis in diabetic conditions as a result of an increased expression of VEGFR1 and a reduction in the level of VEGFR2 (Liu et al. 2013). Conversely, the interaction between VEGF and overexpressed VEGFR2 produces normal angiogenesis and increased cell viability (Liu et al. 2013). Thus, we examined whether Tβ4 reversed the phosphorylation of VEGFR1 and VEGFR2 against AGEs. As shown in Fig.1D, Tβ4 significantly reversed the levels of p-VEGFR2 against AGEs, but did not reduce the level of p-VEGFR1.

Figure 1. Effect of Tβ4 on cellular toxicity in HUVECs incubated with or without AGEs.

Figure 1

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A, cell viability was assessed after incubation with AGEs at various concentrations (100, 200, 300 and 500 μg ml−1) for up to 72 h. B, HUVEC viability was assessed after treatment with 300 μg ml−1 AGEs for 48 h after pretreatment with Tβ4 at various concentrations (0, 0.01, 0.1, 0.3 and 0.5 μg ml−1) for 1 h. C, ROS assay was performed after treatment of HUVECs with 300 μg ml−1 AGEs for 3 h after pretreatment with Tβ4 at various concentrations (0, 0.01, 0.1, 0.3 and 0.5 μg ml−1) for 1 h. D, protein expressions of p-VEGFR1, VEGFR1, p-VEGFR2, and VEGFR2 were measured in HUVECs under corresponding treatments. Data are mean and SEM values (n = 3). *P < 0.05 vs. control; **P < 0.01 vs. control; #P < 0.05 vs. AGEs-only group; ##P < 0.01 vs. AGEs-only group; ###P < 0.001 vs. AGEs-only group.

Tβ4 reduces the expression of both RAGE and the F-actin to G-actin ratio

The expression patterns of RAGE and the F-actin to G-actin ratio, which are markers of actin cytoskeleton homeostasis, were explored by immunoblotting Tβ4-treated HUVECs with or without AGEs for 48 h (Fig.2). Tβ4 treatment significantly reduced the expression of RAGE relative to the control in a dose-dependent manner (Fig.2A). The F-actin to G-actin ratio was analysed to confirm the pattern of actin cytoskeleton induced by Tβ4. As shown in Fig.2B, Tβ4 also decreased the F-actin to G-actin ratio in a dose-dependent manner. Moreover, Tβ4 inhibited AGEs-induced RAGE expression (Fig.2C). Interestingly, AGEs significantly increased the F-actin to G-actin ratio relative to the control condition, while Tβ4 significantly reduced the AGEs-induced increase in the F-actin to G-actin ratio relative to AGEs-treated cells (Fig.2D). These findings suggest that Tβ4 may play a key role in the expression of RAGE involved in the actin cytoskeleton.

Figure 2. Effects of Tβ4 on RAGE and F-actin to G-actin ratio in HUVECs.

Figure 2

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Immunoblotting analysis was conducted to determine the protein expression of RAGE and the F-actin to G-actin ratio. A and B, protein expression of RAGE was measured (A), and F-actin to G-actin ratio was calculated (B) in HUVECs treated with Tβ4 at various concentrations (0.01–0.5 μg ml−1) for 48 h. C and D, protein expression of RAGE was measured (C), and F-actin to G-actin ratio was calculated (D) in HUVECs pretreated with or without 0.3 μg ml−1 Tβ4 for 1 h with 300 μg ml−1 AGEs for 48 h. Data are mean and SEM values (n = 3). *P < 0.05 vs. control; ***P < 0.001 vs. control; ##P < 0.01 vs. AGEs-only group.

RAGE expression was regulated by actin cytoskeleton involved in Tβ4

The relationship between RAGE and Tβ4 was determined using Phalloidin, which binds F-actin and prevents the depolymerization of cells. Phalloidin strongly induces actin polymerization and stabilizes polymerized actin (Wehland et al. 1977). Phalloidin was therefore used to examine the disorganization of the actin cytoskeleton resulting from increases in the F-actin to G-actin ratio, and as an antagonist to the Tβ4-induced remodelling of the actin cytoskeleton. As shown in Fig.3A, Phalloidin-treated cells did not exhibit significant toxicity when it was used at concentrations of up to 100 μm for 48 h. Moreover, Phalloidin did not affect RAGE expression, but strongly increased the F-actin to G-actin ratio in a dose-dependent manner (Fig.3B, C). In a further experiment, 50 μm Phalloidin was used to regulate only the F-actin to G-actin ratio. The expression of RAGE in cells treated with AGEs and Phalloidin was greater than in those treated with AGEs alone, but that of those treated with Phalloidin did not differ significantly from the control condition (Fig.3D). Phalloidin counteracted the attenuation effect of Tβ4 on AGEs-induced RAGE expression (Fig.3D). To clarify the change in the F-actin to G-actin ratio pattern associated with RAGE, we examined the same set of protein samples using Phalloidin. As shown in Fig.3E, Phalloidin significantly counteracted the change in the F-actin to G-actin ratio caused by treatment with Tβ4 and AGEs, consistent with the findings shown in Fig.3D. These results together suggest that the regulation of the F-actin to G-actin ratio by Tβ4 plays a pivotal role in the AGEs-induced expression of RAGE.

Figure 3. Effects of Tβ4 and Phalloidin on RAGE and the F-actin to G-actin ratio in AGEs-treated HUVECs.

Figure 3

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A, cell viability assay to evaluate toxicity of Phalloidin at various concentrations (10, 50 and 100 μm) for 48 h. B and C, the protein expression of RAGE (B) and F-actin to G-actin ratio (C) were calculated in HUVECs treated with Phalloidin for 48 h. D and E, AGEs-treated HUVECs were incubated with or without 0.3 μg ml−1 Tβ4, or 50 μm Phalloidin for 48 h, and then the protein expression of RAGE was measured (D), and F-actin to G-actin ratio was calculated (E). F, protein expressions of p-AKT, AKT, p-GSK3β and GSK3β were measured under the corresponding treatment. Data are mean and SEM values (n = 3). *P < 0.05 vs. control; ***P < 0.001 vs. control; #P < 0.05 vs. AGEs-only group; ##P < 0.01 vs. AGEs-only group; ###P < 0.001 vs. AGEs-only group; +P < 0.05 vs. Tβ4 + AGEs group; +++P < 0.001 vs. Tβ4 + AGEs group.

The AKT-GSK3β signalling pathway, which involves factors related to cell survival and proliferation that are inhibited by the RAGE signalling pathway, was explored to analyse the downstream pathway (Fig.3F). As shown in Fig.3F, p-AKT and p-GSK3β exhibited the same tendencies in each experimental group. AGEs attenuated p-AKT and p-GSK3β, and this effect was reversed by Tβ4. p-AKT and p-GSK3β were also reduced in cells treated with Phalloidin only, possibly caused by its own cellular pathway. The effects of Tβ4 on p-AKT and p-GSK3β were significantly blocked by Phalloidin treatment in AGEs-treated cells. It therefore appears that Tβ4 attenuates RAGE expression via the maintenance of actin cytoskeleton homeostasis.

Tβ4 attenuates the vascular cellular toxicity induced by AGEs via remodelling of the actin cytoskeleton

Cell viability, ROS and VEGFR were analysed to clarify how the effect of Tβ4 on the actin cytoskeleton attenuates AGEs-induced vascular cellular toxicity. As shown in Fig.4A, treatment of cells with Phalloidin and AGEs resulted in a greater degree of toxicity than when they were treated with AGEs alone. Moreover, Phalloidin significantly counteracted the effect of Tβ4 on AGEs-induced vascular cellular toxicity in HUVECs. The level of ROS in cells treated with Phalloidin and AGEs was greater than with AGEs alone (Fig.4B). The ROS assay revealed opposing tendencies compared to cell viability. The attenuation effect of Tβ4 on ROS against AGEs was significantly blocked by Phalloidin.

Figure 4. Effects of Tβ4 and Phalloidin on cellular toxicity in AGEs-treated HUVECs.

Figure 4

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A, cell viability was assessed after incubation with AGEs for 48 h, following pretreatment with 0.3 μg ml−1 Tβ4 or 50 μm Phalloidin for 1 h. B, ROS were assessed in HUVECs after treatment with AGEs for 3 h following pretreatment with Tβ4 or Phalloidin for 1 h. C, protein expressions of p-VEGFR1, VEGFR1, p-VEGFR2 and VEGFR2 were measured in HUVECs under corresponding treatment. Data are the mean and SEM values (n = 3). *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control; #P < 0.05 vs. AGEs-only group; ##P < 0.01 vs. AGEs-only group; ###P < 0.001 vs. AGEs-only group; +P < 0.05 vs. Tβ4 + AGEs group; ++P < 0.01 vs. Tβ4 + AGEs group.

We examined whether Phalloidin counteracts the Tβ4-induced expression of p-VEGFR2 against AGEs (Fig.4C). Phalloidin did not appear to alter the levels of p-VEGFR1 in cells treated with both Tβ4 and AGEs, but it significantly increased levels of p-VEGFR1 in those treated with AGEs alone. This means that the disorganization of the actin cytoskeleton induced by Phalloidin increased p-VEGFR1, which in turn induced abnormal vasculogenic factors. The AGEs-induced reduction of p-VEGFR2 was greater with Tβ4, and Phalloidin significantly attenuated the Tβ4-induced increase in p-VEGFR2. Thus, Tβ4 appears to attenuate the vascular cellular toxicity induced by AGEs via remodelling of the actin cytoskeleton.

Actin cytoskeleton-associated RAGE is affected by intracellular Tβ4

As noted above, external Tβ4 treatment strongly attenuates RAGE expression in HUVECs, as revealed by the change in the F-actin to G-actin ratio. To examine whether intrinsic Tβ4 affects RAGE, Tβ4 siRNA was added to the corresponding treatments (Fig.5). Tβ4 siRNA-treated cells did not exhibit severe toxicity, while the viability of cells treated with both Tβ4 siRNA and AGEs was significantly decreased relative to those treated with AGEs alone (Fig.5A). Tβ4 counteracted the reduced cell viability induced by Tβ4 siRNA in AGEs-treated cells. As shown in Fig.5B, Tβ4 siRNA diminished the expression of Tβ4 and increased that of RAGE. Treatment with AGEs did not alter Tβ4 expression, but increased RAGE expression relative to the control. Moreover, the addition of exogenous Tβ4 to cells treated with both Tβ4 siRNA and AGEs increased the expression of Tβ4, but decreased that of RAGE.

Figure 5. Effects of endogenous intracellular Tβ4 on the cellular toxicity associated with RAGE and the actin cytoskeleton.

Figure 5

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A, cell viability was assessed after HUVECs were treated with AGEs for 48 h with or without Tβ4, Tβ4 siRNA or scrambled siRNA. B and C, the protein expression of RAGE was measured (B), and F-actin to G-actin ratio was calculated (C) in HUVECs under the corresponding treatment. Data are mean and SEM values (n = 3). *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control; #P < 0.05 vs. AGEs-only group; ##P < 0.01 vs. AGEs-only group; ###P < 0.001 vs. AGEs-only group; +P < 0.05 vs. Tβ4 siRNA + AGEs group; ++P < 0.01 vs. Tβ4 siRNA + AGEs group; +++P < 0.001 vs. Tβ4 siRNA + AGEs group.

The F-actin to G-actin ratio pattern was confirmed using the same set of protein samples (Fig.5C). As expected, the F-actin to G-actin ratio pattern exhibited similar tendencies to those of RAGE expression shown in Fig.5B. The extinction of Tβ4 in Tβ4 siRNA-treated cells aggravated the F-actin to G-actin ratio induced by AGEs, representing a hugely disrupted actin cytoskeleton homeostasis. Treatment with exogenous Tβ4 attenuated this crucial alteration of the F-actin to G-actin ratio. These results suggest that intracellular Tβ4 prevents the cellular toxicity induced by the RAGE signalling pathway, resulting in the maintenance of actin cytoskeleton homeostasis.

AGEs attenuate vascular-like tube formation of HUVECs is reversed by Tβ4 via remodelling of actin cytoskeleton

To confirm whether Tβ4 could reverse the inhibition effect of AGEs on the vasculogenic property of HUVECs, we performed a vascular-like tube formation assay on Matrigel, a commonly used method for in vitro vasculogenesis. Cells were seeded on Matrigel in the 96-well culture plate with various conditioned media. After AGEs treatment, the vascular-like tube was developed and photographed at 12 h of incubation (Fig.6A). As previous studies have suggested, Tβ4 accelerated vascular-like tube formation, while AGEs and Phalloidin attenuate structures of tube formation compared with control. Tβ4 significantly counteracted the effect of AGEs and Phalloidin, consistent with previous results. Tube formation with AGEs was significantly attenuated with absence of Tβ4 induced by siRNA (P < 0.01, Fig.6A, panel 6), as well as Phalloidin induction (P < 0.05, Fig.6A, panel 10). The improvement of Tβ4 in AGEs-affected tube formation was attenuated by siRNA or Phalloidin (Fig.6A, panels 8 and 11). Quantitative analysis provided more detail numerical data on the total length of vascular-like tubes (Fig.6B). These results suggest that AGEs attenuate vascular-like tube formation of HUVECs, and that this is reversed by Tβ4 via remodelling of the actin cytoskeleton.

Figure 6. Effect of Tβ4 on vascular-like tube formation of HUVECs.

Figure 6

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A, HUVECs in the presence or absence of Tβ4 induced by siRNA pretreatment were treated with or without Tβ4, AGEs or Phalloidin on to Matrigel for 12 h. Each numbered panel indicates the treatment conditions below (1, control; 2, ccrambled siRNA; 3, Tβ4 siRNA; 4, AGEs; 5, Tβ4; 6, AGEs + Tβ4 siRNA; 7, AGEs + Tβ4; 8, AGEs + Tβ4 + Tβ4 siRNA; 9, Phalloidin; 10, Phalloidin + AGEs; 11, Phalloidin + AGEs + Tβ4). B, the total length of vascular-like tubes was measured and normalized against control. The length of tubes was measured using an Image Pro analysis program. Data are mean and SEM values (n = 3). *P < 0.05 vs. panel 1; ***P < 0.001 vs. panel 1; #P < 0.05 vs. panel 4; ##P < 0.01 vs. panel 4; ###P < 0.001 vs. panel 4; ++P < 0.01 vs. panel 7; +++P < 0.001 vs. panel 7; @@@P < 0.001 vs. panel 9; $$$P < 0.001 vs. panel 10.

Discussion

Intracellular Tβ4 enhances actin cytoskeleton homeostasis, thus protecting the cell from external damage. It has been shown that remodelling of the actin cytoskeleton induced by Tβ4 is pivotal at various stages of angiogenesis (Pirkkala et al. 2001). Actin is a cytoskeleton element that is essential for cell proliferation and motility, both of which are crucial for angiogenesis. Remodelling of actin dynamics leads to the formation of protrusive structures, including lamellipodia and filopodia, and generates the intracellular forces that are required for cell migration (Pollard & Borisy, 2003).

There is considerable evidence that Tβ4 exerts therapeutic effects in diabetic vascular disorders such as peripheral neuropathy (Wang et al. 2012) and peripheral vascular diseases (Treadwell et al. 2012). Indeed, it has been demonstrated that Tβ4 is involved in the protection and recovery from diabetic injury and exerts its properties via anti-apoptotic, anti-inflammatory and angiogenic functions both in vitro and in vivo (Malinda et al. 1999). Tβ4 was recently shown to attenuate hyperglycaemia and improve insulin resistance in diabetic mice (Zhu et al. 2012). However, the effect of Tβ4 on RAGE, which is a critical cause of diabetic injury in patients, has not yet been investigated.

The accumulation of AGEs has been noted as a crucial pathophysiological mechanism in the development of diabetic complications with ageing, and in combination with RAGE exerts deleterious effects (Chen et al. 2012). In healthy humans, serum levels of AGEs of about 0.8–1.4 μg ml−1 have been confirmed (Bansal et al. 2013). Diabetic patients with severe vascular complications have serum levels of AGEs that are 3-fold higher than in healthy humans (about 2.17–4.17 μg ml−1). However, this concentration, based on serum levels in diabetic patients, had no harmful effect on HUVECs in our in vitro experiment, or in those of other researchers. Thus, we used a high concentration of AGEs to analyse how AGEs induce a harmful effect in blood vessel endothelial cells. A high concentration of AGEs in HUVECs leads to rapid and massive vascular toxicity locally. This in vitro toxicity indicates dangerous AGEs accumulation in the human body that can be extrapolated to in vivo experiments. Endothelial dysfunction due to the cellular damage induced by RAGE was reported to be a deleterious factor in diabetic patients (Bosevski & Georgievska-Ismail, 2010). A clinical investigation indicated that the RAGE signalling pathway was associated with endothelial dysfunction in the progression of vascular complications. Moreover, the oxidative stress related to the RAGE response may play a key role in vascular disorders (Huijberts et al. 2008). Several studies have explored the disorganization of the actin cytoskeleton and impaired repair of the endothelial cell membrane associated with RAGE-induced hyperpermeability (Hirose et al. 2010; Xiong et al. 2011). However, the mechanism underlying remodelling of the actin cytoskeleton remains unclear.

In a previous study, we investigated the therapeutic effect of Tβ4 in HUVECs under high glucose conditions associated with its potential IGF-1 signalling pathway (Kim & Kwon, 2014a), and found that treatment with Tβ4 leads to burn wound healing in diabetic mice via acceleration of vasculogenesis (Kim & Kwon, 2014b). Moreover, we found evidence related to the reduction of RAGE expression in dermal tissue lysates. In the present study we have demonstrated for the first time that the association between Tβ4 and the actin cytoskeleton regulates RAGE expression and thus plays a pivotal role in the prevention of AGEs-induced vascular cellular toxicity.

As expected, Tβ4 increased the AGEs-induced attenuation of cell viability (Fig.1B) and decreased AGEs-induced ROS (Fig.1C). These results suggest that treatment with exogenous Tβ4 protects HUVECs against AGEs. We have confirmed the protective effect of Tβ4 against the abnormal vasculogenesis induced by AGEs through a comparative analysis of p-VEGFR1 and p-VEGFR2 (Fig.1C). In combination with AGEs, RAGE could trigger vascular hyperpermeability and abnormal, pathological vasculogenesis by inducing VEGF, and may thus be involved in the pathogenesis of vascular disorders (Liu et al. 2013). VEGF induces the proliferation of endothelial cells and the construction of new blood vessels via its interaction with receptors such as VEGFR1 and VEGFR2 (Kalka et al. 2000; Schwarz et al. 2000). Although AGEs attenuated the activation of VEGFR2, it activated VEGFR1 (Liu et al. 2013). This suggests that AGEs induce abnormal vasculogenesis, as in diabetic retinopathy, via the suppression of VEGFR2. As shown in Fig.1C, Tβ4 significantly increased p-VEGFR2 relative to cells treated with AGEs alone. This Tβ4-induced increase in p-VEGFR1 may be attributable to the ability of Tβ4 to induce the expression of VEGF. As mentioned above, diabetic vascular disorders may be partly attributable to AGEs-induced RAGE. Figure2C confirms that RAGE expression was attenuated by Tβ4 against AGEs. Interestingly, the F-actin to G-actin ratio, which reflects the dynamics of actin cytoskeleton homeostasis, exhibited similar tendencies to those shown in Fig.2C (see Fig.2D). Although immunocytochemistry has shown that F-actin stress fibres are reduced by RAGE (Xiong et al. 2011), our quantification analysis of the F-actin to G-actin ratio by the separation of F-actin and G-actin revealed an increase induced by RAGE in combination with AGEs (Fig.2D). Tβ4 significantly attenuated the AGEs-induced F-actin to G-actin ratio.

We clarified this association between Tβ4 and RAGE using Phalloidin, which induces polymerization of the actin cytoskeleton, including an increment of the F-actin to G-actin ratio (Wehland et al. 1977) and remodelling of the actin cytoskeleton induced by Tβ4. Phalloidin did not appear to be associated with any cellular toxicity or RAGE expression when it was present at a concentration of up to 100 μm, while critical changes in the F-actin to G-actin ratio were observed for all treatment concentrations (Fig.3A–C). As shown in Fig.3D and E, Phalloidin effectively blocked the effects of Tβ4, and thereby increased RAGE, consistent with the change in the F-actin to G-actin ratio. Co-treatment with AGEs and Phalloidin increased RAGE expression compared to only AGEs-treated cells (Fig.3D). This result suggests that the increment in the F-actin to G-actin ratio, which was regarded as reflecting an imbalance between G-actin and F-actin, additionally sensitizes RAGE expression. Moreover, the AKT-GSK3β signalling pathway, which was attenuated by RAGE, was also affected by Phalloidin by interfering with the involvement of Tβ4 (Fig.3F). These results suggest that Tβ4 regulates RAGE via remodelling of the actin cytoskeleton. The additional experiment shown in Fig.4 supports this conclusion.

The association between intrinsic Tβ4 and our defined mechanism was explored to determine whether intrinsic Tβ4 plays the same role as RAGE in the actin cytoskeleton. As shown in Fig.5, Tβ4 siRNA diminished Tβ4 expression but increased RAGE expression. Treatment with AGEs did not change the levels of Tβ4. However, AGEs boosted RAGE expression in cells treated with Tβ4 siRNA. Treatment with exogenous Tβ4 reversed the expression of Tβ4 and RAGE in cells treated with both Tβ4 siRNA and AGEs. Interestingly, the F-actin to G-actin ratio showed the same tendencies as that in RAGE expression and opposing tendencies to that in Tβ4 (Fig.5C). Based on these results, we confirmed the vascular-like tube formation of HUVECs seen with previous experimental conditions (Fig.6). Endothelial cell proliferation and migration are the first important steps to vasculogenesis. Vasculogenesis relates the migration of endothelial cells and their organization into a network of tube-like structures (Mu et al. 2006). Vascular-like tube formation on Matrigel is commonly used for analysis of function to vasculogenesis (Nicosia & Ottinetti, 1990). This evidence of vasculogenesis reflects the results of p-VEGFR1 and p-VEGFR2 induced by Tβ4. Tβ4 alleviated the AGEs-attenuated vascular-like tube formation, as shown by evidence that it is involved in RAGE regulated by actin cytoskeleton balance. These results strongly suggest that Tβ4, which occurs naturally in mammalian cells, plays a pivotal role in the regulation of RAGE by remodelling of the actin cytoskeleton (Fig.7).

Figure 7. Schematic diagram of the mechanism underlying the association between Tβ4 and RAGE.

Figure 7

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Intrinsic Tβ4 maintains the homeostasis of the actin cytoskeleton. Tβ4 inhibits RAGE via this actin cytoskeleton remodelling, and then affects the downstream RAGE signalling pathway. The present results indicate that Tβ4 protects vascular cells against AGEs-induced RAGE.

Several previous studies have indicated that Tβ4 markedly improves peripheral neuropathy under diabetic conditions in vivo (Magharious et al. 2011; Wang et al. 2012). However, these studies could not explain that the critical factor involved in diabetic pathogenesis, such as the effect of Tβ4 on RAGE, using its original functions. The present findings are the first to reveal this hitherto important missing link between Tβ4 and diabetic complications. Moreover, we also show for the first time that changes in the actin cytoskeleton effected by Tβ4 are attributable to alterations in protein receptors on the cellular membrane, such as RAGE. This suggests that changes in the actin cytoskeleton may regulate another protein receptor on the cellular membrane. Further research is required to clarify this issue.

In conclusion, the results of the present study show for the first time that Tβ4 attenuates AGEs-induced RAGE expression in HUVECs, and that it plays a pivotal role in the regulation of RAGE via remodelling of the actin cytoskeleton. Based on our findings, we suggest that exogenous Tβ4 could represent a novel therapeutic strategy for the treatment of chronic diabetic patients afflicted with vascular disorders.

Acknowledgments

We thank Ji-Young Na and Kibbeum Song at the laboratory animal centre for help with immunoblotting.

Glossary

AGEs

advanced glycation end products

GSK

glycogen synthase kinase

HUVEC

human umbilical vein endothelial cell

RAGE

receptor of advanced glycation end products

ROS

reactive oxygen species

Tβ4

thymosin beta 4

VEGFR

vascular endothelial growth factor receptor

Additional information

Competing interests

The authors have no conflict of interest.

Author contributions

S.K. and J.K. both contributed to the conception and design of the study, and contributed to the acquisition, analysis and interpretation of the data. Both authors participated in drafting and revising the article and have approved the final version of the article.

Funding

This work was supported by a grant from the National Research Foundation of Korea funded by the Korean Government (NRF-2012R1A1B3003531).

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