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Liraglutide attenuates the migration of retinal pericytes induced by advanced glycation end products
Peptides 105 (2018) 7–13
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Liraglutide attenuates the migration of retinal pericytes induced by advanced glycation end products
T
Wen-jian Lin, Xue-fei Ma, Ming Hao, Huan-ran Zhou, Xin-yang Yu, Ning Shao, Xin-yuan Gao, ⁎ Hong-yu Kuang Department of Endocrinology, The First Affiliated Hospital of Harbin Medical University, Harbin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Liraglutide Migration Retinal pericytes Glucagon-like peptide-1 receptor Diabetic retinopathy
Retinal pericyte migration represents a novel mechanism of pericyte loss in diabetic retinopathy (DR), which plays a crucial role in the early impairment of the blood-retinal barrier (BRB). Glucagon-like peptide-1 (GLP-1) has been shown to protect the diabetic retina in the early stage of DR; however, the relationship between GLP-1 and retinal pericytes has not been discussed. In this study, advanced glycation end products (AGEs) significantly increased the migration of primary bovine retinal pericytes without influencing cell viability. AGEs also significantly enhanced phosphatidylinositol 3-kinase (PI3K)/Akt activation, and changed the expressions of migration-related proteins, including phosphorylated focal adhesion kinase (p-FAK), matrix metalloproteinase (MMP)-2 and vinculin. PI3K inhibition significantly attenuated the AGEs-induced migration of retinal pericytes and reversed the overexpression of MMP-2. Glucagon-like peptide-1 receptor (Glp1r) was expressed in retinal pericytes, and liraglutide, a GLP-1 analog, significantly attenuated the migration of pericytes by Glp1r and reversed the changes in p-Akt/Akt, p-FAK/FAK, vinculin and MMP-2 levels induced by AGEs, indicating that the protective effect of liraglutide was associated with the PI3K/Akt pathway. These results provided new insights into the mechanism underlying retinal pericyte migration. The early use of liraglutide exerts a potential bebefical effect on regulating pericyte migration, which might contribute to mechanisms that maintain the integrity of vascular barrier and delay the development of DR.
1. Introduction Diabetic retinopathy (DR) is a leading cause of acquired visual impairment worldwide, and the number of patients with DR is estimated to increase to 191.0 million by 2030 [40]. In the early stage of DR, the permeability of the blood-retinal barrier (BRB) increases and the homeostasis of the retinal vasculature is disrupted. Pericytes are specialized cells located at the abluminal surface of capillary vessels, which are essential constituents of the BRB. Vessel permeability directly correlates with the density of pericytes, and in the retina, the pericyte-toendothelial ratio is relatively high (1:1) as compared to other microvascular beds [37], suggesting that pericytes exhibit a superior barrier function in the BRB. The selective loss of pericytes is an early feature of DR pathology, but the underlying mechanism is still controversial. In the animal model of DR, pericyte apoptosis has been detected after > 6 months of hyperglycemia [21]. However, a significant loss of pericytes is already detectable after 3 months of experimentally induced
diabetes [14]. Hammes et al. provided morphological evidence showing that pericyte migration represents a novel mechanism of pericyte loss in the diabetic retina [30], thereby explaining the discrepancy between the total extent of pericyte loss and published data of pericyte apoptosis in the diabetic retina. Currently, studies about the relationship between pericyte migration and blood barriers have primarily focused on brain vessels. Migration represents a potential protective mechanism that prevents pericyte death during injuries [9], but the early detachment of pericytes from endothelial cells in stroke contributes to the lack of close contact between these cells and enhanced the blood-brain barrier leakage, thereby promoting stroke progression [23]. Focal adhesions (FAs) are composed of a high density of proteins and are an integrin-containing protein complex. FAs provide dynamic links between the extracellular matrix (ECM) and intracellular cytoskeleton, and play crucial roles in regulating cell motility. Within the cell, the intracellular domain of integrin binds to the cytoskeleton via adapter proteins such as focal
Abbreviations: AGEs, advanced glycation end products; BRB, blood-retinal barrier; DR, diabetic retinopathy; ECM, extracellular matrix; FAK, focal adhesion kinase; FAs, focal adhesions; GLP-1, glucagon-like peptide-1; Glp1r, glucagon-like peptide-1 receptor; MMP, matrix metalloproteinase; p-FAK, phosphorylated focal adhesion kinase; PI3K, phosphatidylinositol 3kinase ⁎ Corresponding author at: No.23 Youzheng Street, Harbin, Heilongjiang Province, 150000, China. E-mail address: [email protected] (H.-y. Kuang). https://doi.org/10.1016/j.peptides.2018.05.003 Received 14 February 2018; Received in revised form 3 May 2018; Accepted 6 May 2018 Available online 07 May 2018 0196-9781/ © 2018 Elsevier Inc. All rights reserved.
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2.2. Immunofluorescence staining
adhesion kinase (FAK) or vinculin and this integrin-adapter proteincytoskeleton complex forms the basis of FAs. FAK is a non-receptor protein tyrosine kinase that is principally located in the FAs [28]. It is also a key molecule that controls cell migration, due to its involvement in the regulation of FA turnover [36,25]. Vinculin is a linker protein that is specifically associated with FAs and interacts with several other linker proteins involved in such binding interactions. It also binds to βcatenin to stabilize E-cadherin (E-cad) at the cell surface [29]. Therefore, vinculin is responsible for the attachment of actin to the plasma membrane and cell-cell adhesion. Matrix metalloproteinase (MMP)-2 is capable of degrading the structural components of the ECM and nonmatrix proteins; it also plays an important role in facilitating cell migration [6]. Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone secreted by the L cells of the intestine that stimulates insulin secretion and suppresses glucagon release in a glucose-dependent manner [20]. GLP1 exerts various non-glycemic effects on multiple tissues [1], and the activation of the glucagon-like peptide-1 receptor (Glp1r) is one of the most important determinants of GLP-1-induced effects [3]. Exendin (9–39) shares 53% sequence homology with GLP-1, and is often applied as a GLP-1 receptor antagonist in experimental studies [35]. Our group reported the expression of Glp1r in retinal ganglion cells (RGC-5) and analyzed the protective mechanism of GLP-1 under high-glucose conditions [15]. GLP-1 protects the diabetic retina in the early stage of DR [27]; however, the effect of GLP-1 on retinal pericytes has not yet been studied. Liraglutide is a human GLP-1 analog that shares 97% sequence identity with the native hormone, whose half-life is approximately 11–13 h [26]. Liraglutide promotes a homogeneous reduction in glycemia in the clinical treatment of diabetes, and its pleiotropic protective effects on diabetic complications have also received increasing attentions [17,24,16]. GLP-1 modulates the migration of vascular smooth muscle cells, cancer cells, lymphocytes, and mesenchymal stem cells [38,42,22,44], and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is mostly studied in these studies. However, the underlying mechanism is not clear. PI3K-mediated signal transduction regulates the migration of many different cell types and contributes to various aspects of cell migration [2]. LY294002, a broad-spectrum PI3K inhibitor, is often applied in PI3K/Akt signaling studies. Advanced glycation end products (AGEs) represent non-enzymatic glycosylation products of cellular proteins formed during hyperglycemia. The accumulation of AGEs is implicated in several diabetic complications and is critical for the pathogenesis of DR [39]. In addition, AGEs induce the migration of many types of cells [32,43,8]. The accumulation of AGEs is a prolonged process, and hence, we speculated that in retinal pericyte cultures, AGEs would induce cell migration in vitro before influencing cell viability, and liraglutide would participate in this process by exerting protective effects.
Pericytes were seeded in 24-well plates. Briefly, cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 20 min. After an incubation with primary antibodies against Glp1r (1:100 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), α-smooth muscle actin (1:100 dilution, Abcam, Cambridge, UK), glial fibrillary acidic protein (1:100 dilution, Santa Cruz Biotechnology), and factor VIII (1:100 dilution, Santa Cruz Biotechnology) at 4 °C overnight, cells were incubated with secondary antibodies (1:200, ZSGB-BIO, Beijing, China) at 37 °C for 1 h, and 4′6diamino-2-phenylindole (Beyotime, Shanghai, China) was added to stain the cell nuclei. Finally, cells were analyzed under a fluorescence microscope (EVOS FL Auto, Life Technologies). 2.3. Measurement of cell viability Cell viability was detected using the CCK-8 kit (Dojindo, Shanghai, China). Pericytes were seeded in a 96-well microplate at a density of 5000 cells/well and treated with AGE-BSA (0, 25, 50, 100, or 150 μg/ mL) and Control-BSA for 24 h or treated with liraglutide (0, 1, 10, 25, 50, or 100 nM) for 24 h. Subsequently, the CCK-8 reagent (10 μL/well) was added to each well, and the plate was incubated at 37 °C for 3 h. A monochromator microplate reader was used to count the viable cells by measuring the absorbance at 450 nm. 2.4. Transwell assay Pericytes were suspended in serum-free medium and seeded (2 × 104) in the upper compartment of the transwell chambers (Corning, Manassas, VA, USA), whereas the lower compartment was filled with complete medium. Intervention reagents were added to both the upper and lower chambers. After a 24 h incubation at 37 °C in the presence of 5% CO2, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Cells on the upper surface of the filter were carefully removed with a cotton swab. The cells that had migrated to the lower surface were counted in six high-power random fields from three independent experiments using a microscope that assessed the average number of migrating cells. 2.5. Wound-healing assay Pericytes were grown to complete confluency in 6-well plates, followed by treatment with 10 μg/mL mitomycin C for 2 h. A wound was created by scratching the length of the well with a sterile pipette tip (10 μL), and then the cells were incubated with intervention reagents for 24 h. Images of the wound areas were acquired at 0 and 24 h postwounding using a microscope. The percentage of wound closure was estimated using Image-Pro Plus software. Each assay was repeated 3 times. The percentage of wound healing was calculated using the following equation: [1 − (empty area X h/empty area 0 h)] × 100 [4].
2. Materials and methods 2.1. Cell culture
2.6. Western blot analysis Primary fetal bovine retinal pericytes were isolated as described previously [31]. Pericytes were cultured in DMEM containing 20% FBS (ScienCell, San Diego, CA, USA) in a 5% CO2 humidified atmosphere at 37 °C. Cells were used at passages 3–5 in subsequent experiments. Cells in some groups were pretreated with LY294002 (10 μM, Sigma-Aldrich, St Louis, MO, USA) or liraglutide (Novo Nordisk, Copenhagen, Denmark) for 2 h, or pretreated with a GLP-1 receptor antagonist [exendin (9–39), 100 nM, Sigma-Aldrich] for 2.5 h, followed by exposure to Control-BSA (Biovision, San Francisco, CA, USA) or AGE-BSA (Biovision) for 24 h, exendin (9–39) was added 30 min before the addition of liraglutide. The standardized product AGE-BSA is considered to represent AGEs and was used throughout this study.
Equivalent amounts of proteins were resolved on SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking for 1 h with 5% milk, the membranes were incubated overnight at 4 °C with the following primary antibodies: Glp1r (1:1000, Santa Cruz Biotechnology), Akt (1:1000, CST, Danvers, MA, USA), p-Akt (1:1000, CST), FAK (1:1000, CST), phosphorylated focal adhesion kinase (pFAK397, 1:1000, Abcam), vinculin (1:1000, Santa Cruz Biotechnology), MMP-2 (1:1000, Abcam), and β-actin (1:2000, ZSGB-BIO). Subsequent incubation with horseradish peroxidase-conjugated secondary antibody (1:2000, ZSGB-BIO) was performed, and the bands were then visualized with the Molecular Imaging System (Bio-Rad, USA). 8
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Fig. 1. Indentification of retinal pericytes and an examination of cell viability. (A) Primary bovine retinal pericytes were identified by positive staining for α-smooth muscle actin (green). (B) Cells were treated with ControlBSA (control group) or different concentrations of AGE-BSA (0, 25, 50, 100 or 150 μg/mL) for 24 h, followed by the detection of cell viability using the CCK-8 kit. Treatment with AGE-BSA at concentrations ≤ 100 μg/mL did not significantly alter cell viability. Scale bar = 50 μm. Data were reported as mean ± SEM for three independent experiments (*P < 0.05 vs control group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. AGE-BSA induced retinal pericyte migration. Pericytes were treated with Control-BSA (100 μg/mL) or different concentrations of AGE-BSA (25, 50 or 100 μg/ mL) for 24 h. (A–D) The results of transwell (scale bar = 200 μm) and wound-healing (10× objective lens) assays showed that AGE-BSA (100 μg/mL) induced cell migration. (E–F) AGE-BSA (100 μg/mL) altered the levels of p-FAK/FAK, MMP-2 and vinculin. Data were reported as mean ± SEM for three independent experiments.**P < 0.01 vs control group, ***P < 0.001 vs control group; P was calculated using Student’s t-test; A.U., arbitrary units.
2.7. Statistical analysis
3. Results
The values were represented as means ± SEM from three independent experiments. Statistical analysis was processed via one-way ANOVA, followed by Turkey’s post hoc test or Student’s t-test. GraphPad Software was used for all the statistical analysis, P < 0.05 was considered as statistically significant.
3.1. AGE-BSA induced retinal pericyte migration Primary bovine retinal pericytes were identified by positive staining for α-smooth muscle actin (Fig. 1A) and negative staining for glial fibrillary acidic protein and factor VIII (data not shown). Next, we 9
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Fig. 3. The inhibition of PI3K/Akt signaling attenuated the AGE-BSA-induced migration of retinal pericytes. (A–B) AGE-BSA increased the ratio of p-Akt/Akt levels. (C–F) According to the results of transwell (scale bar = 200 μm) and wound-healing (10× objective lens) assays, LY294002 decreased the AGE-BSA-induced migration of retinal pericytes. (G–H) LY294002 reversed the overexpression of MMP-2 and increase in p-Akt/Akt levels induced by AGE-BSA, but did not alter the levels of p-FAK/FAK and vinculin. Data were reported as mean ± SEM for three independent experiments. *P < 0.05 vs control group, **P < 0.01 vs control group, *** P < 0.001 vs control group; #P < 0.05 vs AGE-BSA group, ###P < 0.001 vs AGE-BSA group, ★P < 0.05 vs AGE-BSA group; P was calculated using Student’s ttest; A.U., arbitrary units.
was not statistically significant. Treatment with 150 μg/mL AGE-BSA significantly decreased cell viability compared with the control group. We wanted to examine cell migration after excluding an impact of cell viability; therefore, we chose AGE-BSA concentrations that did not exceed 100 μg/mL in the subsequent studies. AGE-BSA induced retinal pericyte migration in a dose-dependent
examined the viability of retinal pericytes that had been treated with Control-BSA or different concentrations of AGE-BSA (0, 25, 50, 100, or 150 μg/mL) for 24 h. Control-BSA did not alter the cell viability (Fig. 1B), and hence, it was used as the control in the subsequent studies. Control-BSA and low concentrations of AGE-BSA slightly increased the cell viability compared with the blank control, but the difference 10
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Fig. 4. Glp1r expression in retinal pericytes and an examination of cell viability. (A) Image of immunofluorescence staining for Glp1r (green) in retinal pericytes. (B) Western blot showing Glp1r expression in retinal pericytes; RIN cells served as the positive control. (C) Cells were treated with different concentrations of liraglutide (0, 1, 10, 25, 50 or 100 nM) for 24 h, and then cell viability was assessed using the CCK-8 kit. Liraglutide did not affect cell viability at the concentrations employed in this experoment. Scale bar = 50 μm. Data were reported as mean ± SEM for three independent experiments (*P < 0.05 vs control group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
subsequent studies was 10 nM, which was similar to the estimated maximal concentration for a single subcutaneous dose of 0.6 mg/day (product monograph for liraglutide, Victoza). Furthermore, we explored whether liraglutide regulated cell migration. After a 24 h incubation, liraglutide attenuated the AGE-BSAinduced migration of retinal pericytes in transwell and wound-healing assays, and exendin (9–39) (100 nM) suppressed the protective effect of liraglutide (Fig. 5A and C). The number of migrating cells and the percentage of wounded healing area in AGE-BSA + liraglutide group were significantly decreased compared with the AGE-BSA group, whereas in the AGE-BSA + liraglutide + exendin (9–39) group, the two indicators were significantly increased by 1.66- and 5.04-fold compared with the AGE-BSA + liraglutide group, respectively (Fig. 5B and D). Compareing AGE-BSA + liraglutide group with AGE-BSA group, western blots showed that liraglutide significantly reduced PI3K/Akt activation, reversed the increased levels of MMP-2 and p-FAK/FAK, and significantly upregulated the low level of vinculin induced by the AGEBSA treatment (Fig. 5E and F).
manner in both transwell (Fig. 2A) and wound-healing assays (Fig. 2C). However, a statistical significant difference was not observed until the concentration of AGE-BSA was increased to 100 μg/mL. At this concentration, AGE-BSA significantly enhanced the migration of retinal pericytes by 1.84- and 6.46-fold in the two assays compared with the control group, respectively (Fig. 2B and D). Vinculin, MMP-2, and pFAK/FAK play vital roles in cell migration. Thus, we examined whether their levels were altered during AGE-BSA-mediated migration. After a 24 h incubation, 100 μg/mL AGE-BSA significantly decreased vinculin expression by 1.62-fold, and increased the levels of MMP-2 and p-FAK/ FAK in retinal pericytes by 1.37- and 1.79-fold compared with the control group, respectively (Fig. 2E and F). 3.2. The PI3K/Akt pathway was involved in AGE-BSA-induced retinal pericyte migration AGE-BSA induced the activation of the PI3K/Akt pathway in a dosedependent manner. Treatment with 100 μg/mL AGE-BSA increased the phosphorylation of Akt 1.38-fold compared with the control group (Fig. 3A and B). Next, we explored whether PI3K/Akt pathway regulated the migration of retinal pericytes. We pretreated the cells with the PI3K inhibitor, LY294002 (10 μM) for 2 h, followed by Control-BSA (100 μg/mL) or AGE-BSA (100 μg/mL) for 24 h. As shown in the images from the transwell and wound-healing assays (Fig. 3C and E), LY294002 attenuated the AGE-BSA-induced migration of retinal pericytes and significantly decreased the number of migrating cells and the percentage of the wounded healing area compared with the AGE-BSA group (Fig. 3D and F). Western blot analyses further revealed that the activation of PI3K was significantly suppressed in AGEBSA + LY294002 group compared with the AGE-BSA group, and PI3K inhibition reversed the overexpression of MMP-2; however, it did not affect the levels of p-FAK/FAK and vinculin (Fig. 3G and H).
4. Discussion Pericyte migration is an alternative or additional mechanism underlying pericyte loss in diabetic retinopathy [30], which plays a crucial role in the early impairment of the BRB and the increase in vessel permeability. In the pathogenesis of DR, the loss of pericytes occurs earlier than the formation of new capillaries. However, previous studies of vascular cell migration have primarily focused on endothelial cells and angiogenesis. A large number of AGE-BSA could induce cell apoptosis, and hence, we selected the AGE-BSA concentrations that did not decrease cell viability to examine the migration of retinal pericytes. The intervention was applied for 24 h, and thus did not produce longterm damage. Based on the results from the current study, AGE-BSA increased the migration of retinal pericytes without influencing cell viability, which might be a self-protective behavior to avoid further injuries to the cell. However, the migration of pericytes contributes to increased microvascular permeability and promotes the progression of DR. At the ultrastructural level, the separation of brain pericytes from brain microvessels has been observed at as early as 2 h after the onset of hypoxia [13]. Pericyte migration from the basement membrane has also been observed after 1 h of irreversible ischemia [10]. Thus, we speculated that retinal pericyte migration might be an early stress response to external noxious stimuli. Pericytes migrate from vessels into the perivascular parenchyma, disrupt the integrity of BRB, and further aggravate the vascular injuries. Migration is a highly integrated process that required sophisticated
3.3. Liraglutide attenuated AGE-BSA-induced retinal pericytes migration and PI3K activation We detected the expression of Glp1r in retinal pericytes using immunofluorescence staining (green, Fig. 4A). In addition, the western blot assay also revealed the expression of Glp1r in retinal pericytes; rat insulinoma cells (RIN-m5F cells) were used as the positive control (Fig. 4B). Next, cells were cultured with different concentrations of liraglutide (1, 10, 25, 50, or 100 nM) for 24 h to evaluate the cellular toxicity. Treatments with up to 100 nM liraglutide were tolerated by retinal pericytes, without a considerable effect on cell survival (Fig. 4C). Additionally, the concentration of liraglutide utilized in 11
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Fig 5. Liraglutide attenuated the AGE-BSA-induced migration of retinal pericytes. (A–D) The results of the transwell (scale bar = 200 μm) and wound-healing (10× objective lens) assays showed that liraglutide attenuated the AGE-BSA-induced migration of retinal pericytes, and exendin (9–39) suppressed the effect of liraglutide. (E–F) Liraglutide reversed the AGE-BSA-induced changes in the ratio of p-Akt/Akt levels, decreased the MMP-2 and p-FAK/FAK levels, and increased the low vinculin levels. Data were reported as mean ± SEM for three independent experiments. *P < 0.05 vs control group, **P < 0.01 vs control group, ***P < 0.001 vs control group; #P < 0.05 vs AGE-BSA group, ##P < 0.01 vs AGE-BSA group, ###P < 0.001 vs AGE-BSA group; ***P < 0.001 vs AGE-BSA + liraglutide group; P was calculated using Student’s t-test; A.U., arbitrary units.
expression decreased following treatment with AGE-BSA. MMP-2 degrades ECM components, thereby facilitating cell migration. For instance, the cleavage of laminin-5 by MMP-2 reveals a cryptic site that enhances endothelial cell migration [11]. Here, AGE-BSA simultaneously increased the migration of retinal pericytes and the expression of intracellular MMP-2, further confirming that MMP-2 also participates in promoting cell motility. In the present study, we detected the expression of Glp1r in retinal pericytes and proved that liraglutide attenuated the AGE-BSA-induced migration of retinal pericytes. GLP-1 has been reported to regulate the migration of several types of cells through the PI3K/Akt signaling pathway [38,42]. Based on the current data, the PI3K inhibitor LY294002 significantly attenuated the AGE-BSA-induced migration of retinal pericytes, and this effect was associated with the inhibition of MMP-2. The pTyr-397 site on FAK modulates the SH2 domains in several cellular signaling proteins, including PI3K [5]. Here, we found that PI3K inhibition did not influence FAK phosphorylation, thereby indicating that PI3K may be activated downstream of FAK.
regulation of structural networks and signaling throughout the cell. Among these, the leading networks include integrin-based FAs, through which the cells engage in adhesive contacts with the surrounding ECM [19]. FAK has emerged as a key signaling component at FAs since its discovery [41]. Over the past 20 years, the regulation of cell migration by integrin signaling through FAK has been well-established in many cell types [25,12]. In the active state, the amino-terminal FERM domain of FAK is displaced by an activating protein [34]. FAK then undergoes autophosphorylation at Tyr-397, in turn recruiting the Src family PTKs and resulting in the phosphorylation of Tyr-576 and Tyr-577 in the FAK activation loop and full FAK activation of the catalytic domain. In the present study, we detected the extent of FAK phosphorylation in pericytes treated with AGE-BSA and observed increased phosphorylation at Try-397, consistent with the elevated migration rate. Vinculin binds to talin and actin as an FA adaptor protein [18]. In response to the increased load, vinculin is recruited to FAs [33], and the enhanced talinvinculin-actin linkages are proposed as critical features required to strengthen adhesions [7]. The current data showed that vinculin
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Pretreatment with liraglutide attenuated the AGE-BSA-induced migration of retinal pericytes, and this effect was attenuated by the GLP-1 receptor antagonist, indicating that liraglutide exerts its protective effect via Glp1r. Thus, this phenomenon may be associated with the simultaneous inhibition of FAK phosphorylation and the suppression of PI3K/Akt activation.
[16]
[17]
5. Conclusion [18]
In summary, we postulate that retinal pericyte migration might be a self-protective behavior to avoid further cell injuries. However, the migration may contribute to increased microvascular permeability and promote the progression of DR. Moreover, we also observed the expression of Glp1r in retinal pericytes and confirmed the protective effect of liraglutide on attenuating of AGE-BSA-induced pericyte migration through Glp1r. We also confirmed that the PI3K/Akt pathway is implicated in this process. Taken together, our results provided novel insights into the mechanism underlying retinal pericyte migration, and the early use of liraglutide may modulate the migration of these cells to maintain the integrity of the BRB.
[19]
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Conflicts of interest [25]
The authors declare no conflict of interest. [26]
Acknowledgment
[27]
This work was supported by the National Natural Science Foundation of China (81670739).
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Carragher, E. Avizienyte, J. Evans, V.G. Brunton, M.C. Frame, The role of focal-adhesion kinase in cancer-a new therapeutic opportunity, Nat. Rev. Cancer 5 (2005) 505–515. J.J. Meier, GLP-1 receptor agonists for individualized treatment of type 2 diabetes mellitus, Nat. Rev. Endocrinol. 8 (2012) 728–742. B. Pang, H. Zhou, H. Kuang, The potential benefits of glucagon-like peptide-1 receptor agonists for diabetic retinopathy, Peptides 100 (2018) 123–126. J.T. Parsons, Focal Adhesion kinase: the first ten years, J. Cell Sci. 116 (2003) 1409–1416. X. Peng, L.E. Cuff, C.D. Lawton, K.A. DeMali, Vinculin regulates cell-surface Ecadherin expression by binding to beta-catenin, J. Cell Sci. 123 (2010) 567–577. F. Pfister, Y. Feng, F. vom Hagen, S. Hoffmann, G. Molema, J.L. Hillebrands, M. Shani, U. Deutsch, H.P. Hammes, Pericyte migration: a novel mechanism of pericyte loss in experimental diabetic retinopathy, Diabetes 57 (2008) 2495–2502. V.A. Primo, J.F. 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Liraglutide attenuates the migration of retinal pericytes induced by advanced glycation end products
T
Wen-jian Lin, Xue-fei Ma, Ming Hao, Huan-ran Zhou, Xin-yang Yu, Ning Shao, Xin-yuan Gao, ⁎ Hong-yu Kuang Department of Endocrinology, The First Affiliated Hospital of Harbin Medical University, Harbin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Liraglutide Migration Retinal pericytes Glucagon-like peptide-1 receptor Diabetic retinopathy
Retinal pericyte migration represents a novel mechanism of pericyte loss in diabetic retinopathy (DR), which plays a crucial role in the early impairment of the blood-retinal barrier (BRB). Glucagon-like peptide-1 (GLP-1) has been shown to protect the diabetic retina in the early stage of DR; however, the relationship between GLP-1 and retinal pericytes has not been discussed. In this study, advanced glycation end products (AGEs) significantly increased the migration of primary bovine retinal pericytes without influencing cell viability. AGEs also significantly enhanced phosphatidylinositol 3-kinase (PI3K)/Akt activation, and changed the expressions of migration-related proteins, including phosphorylated focal adhesion kinase (p-FAK), matrix metalloproteinase (MMP)-2 and vinculin. PI3K inhibition significantly attenuated the AGEs-induced migration of retinal pericytes and reversed the overexpression of MMP-2. Glucagon-like peptide-1 receptor (Glp1r) was expressed in retinal pericytes, and liraglutide, a GLP-1 analog, significantly attenuated the migration of pericytes by Glp1r and reversed the changes in p-Akt/Akt, p-FAK/FAK, vinculin and MMP-2 levels induced by AGEs, indicating that the protective effect of liraglutide was associated with the PI3K/Akt pathway. These results provided new insights into the mechanism underlying retinal pericyte migration. The early use of liraglutide exerts a potential bebefical effect on regulating pericyte migration, which might contribute to mechanisms that maintain the integrity of vascular barrier and delay the development of DR.
1. Introduction Diabetic retinopathy (DR) is a leading cause of acquired visual impairment worldwide, and the number of patients with DR is estimated to increase to 191.0 million by 2030 [40]. In the early stage of DR, the permeability of the blood-retinal barrier (BRB) increases and the homeostasis of the retinal vasculature is disrupted. Pericytes are specialized cells located at the abluminal surface of capillary vessels, which are essential constituents of the BRB. Vessel permeability directly correlates with the density of pericytes, and in the retina, the pericyte-toendothelial ratio is relatively high (1:1) as compared to other microvascular beds [37], suggesting that pericytes exhibit a superior barrier function in the BRB. The selective loss of pericytes is an early feature of DR pathology, but the underlying mechanism is still controversial. In the animal model of DR, pericyte apoptosis has been detected after > 6 months of hyperglycemia [21]. However, a significant loss of pericytes is already detectable after 3 months of experimentally induced
diabetes [14]. Hammes et al. provided morphological evidence showing that pericyte migration represents a novel mechanism of pericyte loss in the diabetic retina [30], thereby explaining the discrepancy between the total extent of pericyte loss and published data of pericyte apoptosis in the diabetic retina. Currently, studies about the relationship between pericyte migration and blood barriers have primarily focused on brain vessels. Migration represents a potential protective mechanism that prevents pericyte death during injuries [9], but the early detachment of pericytes from endothelial cells in stroke contributes to the lack of close contact between these cells and enhanced the blood-brain barrier leakage, thereby promoting stroke progression [23]. Focal adhesions (FAs) are composed of a high density of proteins and are an integrin-containing protein complex. FAs provide dynamic links between the extracellular matrix (ECM) and intracellular cytoskeleton, and play crucial roles in regulating cell motility. Within the cell, the intracellular domain of integrin binds to the cytoskeleton via adapter proteins such as focal
Abbreviations: AGEs, advanced glycation end products; BRB, blood-retinal barrier; DR, diabetic retinopathy; ECM, extracellular matrix; FAK, focal adhesion kinase; FAs, focal adhesions; GLP-1, glucagon-like peptide-1; Glp1r, glucagon-like peptide-1 receptor; MMP, matrix metalloproteinase; p-FAK, phosphorylated focal adhesion kinase; PI3K, phosphatidylinositol 3kinase ⁎ Corresponding author at: No.23 Youzheng Street, Harbin, Heilongjiang Province, 150000, China. E-mail address: [email protected] (H.-y. Kuang). https://doi.org/10.1016/j.peptides.2018.05.003 Received 14 February 2018; Received in revised form 3 May 2018; Accepted 6 May 2018 Available online 07 May 2018 0196-9781/ © 2018 Elsevier Inc. All rights reserved.
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2.2. Immunofluorescence staining
adhesion kinase (FAK) or vinculin and this integrin-adapter proteincytoskeleton complex forms the basis of FAs. FAK is a non-receptor protein tyrosine kinase that is principally located in the FAs [28]. It is also a key molecule that controls cell migration, due to its involvement in the regulation of FA turnover [36,25]. Vinculin is a linker protein that is specifically associated with FAs and interacts with several other linker proteins involved in such binding interactions. It also binds to βcatenin to stabilize E-cadherin (E-cad) at the cell surface [29]. Therefore, vinculin is responsible for the attachment of actin to the plasma membrane and cell-cell adhesion. Matrix metalloproteinase (MMP)-2 is capable of degrading the structural components of the ECM and nonmatrix proteins; it also plays an important role in facilitating cell migration [6]. Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone secreted by the L cells of the intestine that stimulates insulin secretion and suppresses glucagon release in a glucose-dependent manner [20]. GLP1 exerts various non-glycemic effects on multiple tissues [1], and the activation of the glucagon-like peptide-1 receptor (Glp1r) is one of the most important determinants of GLP-1-induced effects [3]. Exendin (9–39) shares 53% sequence homology with GLP-1, and is often applied as a GLP-1 receptor antagonist in experimental studies [35]. Our group reported the expression of Glp1r in retinal ganglion cells (RGC-5) and analyzed the protective mechanism of GLP-1 under high-glucose conditions [15]. GLP-1 protects the diabetic retina in the early stage of DR [27]; however, the effect of GLP-1 on retinal pericytes has not yet been studied. Liraglutide is a human GLP-1 analog that shares 97% sequence identity with the native hormone, whose half-life is approximately 11–13 h [26]. Liraglutide promotes a homogeneous reduction in glycemia in the clinical treatment of diabetes, and its pleiotropic protective effects on diabetic complications have also received increasing attentions [17,24,16]. GLP-1 modulates the migration of vascular smooth muscle cells, cancer cells, lymphocytes, and mesenchymal stem cells [38,42,22,44], and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is mostly studied in these studies. However, the underlying mechanism is not clear. PI3K-mediated signal transduction regulates the migration of many different cell types and contributes to various aspects of cell migration [2]. LY294002, a broad-spectrum PI3K inhibitor, is often applied in PI3K/Akt signaling studies. Advanced glycation end products (AGEs) represent non-enzymatic glycosylation products of cellular proteins formed during hyperglycemia. The accumulation of AGEs is implicated in several diabetic complications and is critical for the pathogenesis of DR [39]. In addition, AGEs induce the migration of many types of cells [32,43,8]. The accumulation of AGEs is a prolonged process, and hence, we speculated that in retinal pericyte cultures, AGEs would induce cell migration in vitro before influencing cell viability, and liraglutide would participate in this process by exerting protective effects.
Pericytes were seeded in 24-well plates. Briefly, cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 20 min. After an incubation with primary antibodies against Glp1r (1:100 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), α-smooth muscle actin (1:100 dilution, Abcam, Cambridge, UK), glial fibrillary acidic protein (1:100 dilution, Santa Cruz Biotechnology), and factor VIII (1:100 dilution, Santa Cruz Biotechnology) at 4 °C overnight, cells were incubated with secondary antibodies (1:200, ZSGB-BIO, Beijing, China) at 37 °C for 1 h, and 4′6diamino-2-phenylindole (Beyotime, Shanghai, China) was added to stain the cell nuclei. Finally, cells were analyzed under a fluorescence microscope (EVOS FL Auto, Life Technologies). 2.3. Measurement of cell viability Cell viability was detected using the CCK-8 kit (Dojindo, Shanghai, China). Pericytes were seeded in a 96-well microplate at a density of 5000 cells/well and treated with AGE-BSA (0, 25, 50, 100, or 150 μg/ mL) and Control-BSA for 24 h or treated with liraglutide (0, 1, 10, 25, 50, or 100 nM) for 24 h. Subsequently, the CCK-8 reagent (10 μL/well) was added to each well, and the plate was incubated at 37 °C for 3 h. A monochromator microplate reader was used to count the viable cells by measuring the absorbance at 450 nm. 2.4. Transwell assay Pericytes were suspended in serum-free medium and seeded (2 × 104) in the upper compartment of the transwell chambers (Corning, Manassas, VA, USA), whereas the lower compartment was filled with complete medium. Intervention reagents were added to both the upper and lower chambers. After a 24 h incubation at 37 °C in the presence of 5% CO2, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Cells on the upper surface of the filter were carefully removed with a cotton swab. The cells that had migrated to the lower surface were counted in six high-power random fields from three independent experiments using a microscope that assessed the average number of migrating cells. 2.5. Wound-healing assay Pericytes were grown to complete confluency in 6-well plates, followed by treatment with 10 μg/mL mitomycin C for 2 h. A wound was created by scratching the length of the well with a sterile pipette tip (10 μL), and then the cells were incubated with intervention reagents for 24 h. Images of the wound areas were acquired at 0 and 24 h postwounding using a microscope. The percentage of wound closure was estimated using Image-Pro Plus software. Each assay was repeated 3 times. The percentage of wound healing was calculated using the following equation: [1 − (empty area X h/empty area 0 h)] × 100 [4].
2. Materials and methods 2.1. Cell culture
2.6. Western blot analysis Primary fetal bovine retinal pericytes were isolated as described previously [31]. Pericytes were cultured in DMEM containing 20% FBS (ScienCell, San Diego, CA, USA) in a 5% CO2 humidified atmosphere at 37 °C. Cells were used at passages 3–5 in subsequent experiments. Cells in some groups were pretreated with LY294002 (10 μM, Sigma-Aldrich, St Louis, MO, USA) or liraglutide (Novo Nordisk, Copenhagen, Denmark) for 2 h, or pretreated with a GLP-1 receptor antagonist [exendin (9–39), 100 nM, Sigma-Aldrich] for 2.5 h, followed by exposure to Control-BSA (Biovision, San Francisco, CA, USA) or AGE-BSA (Biovision) for 24 h, exendin (9–39) was added 30 min before the addition of liraglutide. The standardized product AGE-BSA is considered to represent AGEs and was used throughout this study.
Equivalent amounts of proteins were resolved on SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking for 1 h with 5% milk, the membranes were incubated overnight at 4 °C with the following primary antibodies: Glp1r (1:1000, Santa Cruz Biotechnology), Akt (1:1000, CST, Danvers, MA, USA), p-Akt (1:1000, CST), FAK (1:1000, CST), phosphorylated focal adhesion kinase (pFAK397, 1:1000, Abcam), vinculin (1:1000, Santa Cruz Biotechnology), MMP-2 (1:1000, Abcam), and β-actin (1:2000, ZSGB-BIO). Subsequent incubation with horseradish peroxidase-conjugated secondary antibody (1:2000, ZSGB-BIO) was performed, and the bands were then visualized with the Molecular Imaging System (Bio-Rad, USA). 8
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Fig. 1. Indentification of retinal pericytes and an examination of cell viability. (A) Primary bovine retinal pericytes were identified by positive staining for α-smooth muscle actin (green). (B) Cells were treated with ControlBSA (control group) or different concentrations of AGE-BSA (0, 25, 50, 100 or 150 μg/mL) for 24 h, followed by the detection of cell viability using the CCK-8 kit. Treatment with AGE-BSA at concentrations ≤ 100 μg/mL did not significantly alter cell viability. Scale bar = 50 μm. Data were reported as mean ± SEM for three independent experiments (*P < 0.05 vs control group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. AGE-BSA induced retinal pericyte migration. Pericytes were treated with Control-BSA (100 μg/mL) or different concentrations of AGE-BSA (25, 50 or 100 μg/ mL) for 24 h. (A–D) The results of transwell (scale bar = 200 μm) and wound-healing (10× objective lens) assays showed that AGE-BSA (100 μg/mL) induced cell migration. (E–F) AGE-BSA (100 μg/mL) altered the levels of p-FAK/FAK, MMP-2 and vinculin. Data were reported as mean ± SEM for three independent experiments.**P < 0.01 vs control group, ***P < 0.001 vs control group; P was calculated using Student’s t-test; A.U., arbitrary units.
2.7. Statistical analysis
3. Results
The values were represented as means ± SEM from three independent experiments. Statistical analysis was processed via one-way ANOVA, followed by Turkey’s post hoc test or Student’s t-test. GraphPad Software was used for all the statistical analysis, P < 0.05 was considered as statistically significant.
3.1. AGE-BSA induced retinal pericyte migration Primary bovine retinal pericytes were identified by positive staining for α-smooth muscle actin (Fig. 1A) and negative staining for glial fibrillary acidic protein and factor VIII (data not shown). Next, we 9
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Fig. 3. The inhibition of PI3K/Akt signaling attenuated the AGE-BSA-induced migration of retinal pericytes. (A–B) AGE-BSA increased the ratio of p-Akt/Akt levels. (C–F) According to the results of transwell (scale bar = 200 μm) and wound-healing (10× objective lens) assays, LY294002 decreased the AGE-BSA-induced migration of retinal pericytes. (G–H) LY294002 reversed the overexpression of MMP-2 and increase in p-Akt/Akt levels induced by AGE-BSA, but did not alter the levels of p-FAK/FAK and vinculin. Data were reported as mean ± SEM for three independent experiments. *P < 0.05 vs control group, **P < 0.01 vs control group, *** P < 0.001 vs control group; #P < 0.05 vs AGE-BSA group, ###P < 0.001 vs AGE-BSA group, ★P < 0.05 vs AGE-BSA group; P was calculated using Student’s ttest; A.U., arbitrary units.
was not statistically significant. Treatment with 150 μg/mL AGE-BSA significantly decreased cell viability compared with the control group. We wanted to examine cell migration after excluding an impact of cell viability; therefore, we chose AGE-BSA concentrations that did not exceed 100 μg/mL in the subsequent studies. AGE-BSA induced retinal pericyte migration in a dose-dependent
examined the viability of retinal pericytes that had been treated with Control-BSA or different concentrations of AGE-BSA (0, 25, 50, 100, or 150 μg/mL) for 24 h. Control-BSA did not alter the cell viability (Fig. 1B), and hence, it was used as the control in the subsequent studies. Control-BSA and low concentrations of AGE-BSA slightly increased the cell viability compared with the blank control, but the difference 10
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Fig. 4. Glp1r expression in retinal pericytes and an examination of cell viability. (A) Image of immunofluorescence staining for Glp1r (green) in retinal pericytes. (B) Western blot showing Glp1r expression in retinal pericytes; RIN cells served as the positive control. (C) Cells were treated with different concentrations of liraglutide (0, 1, 10, 25, 50 or 100 nM) for 24 h, and then cell viability was assessed using the CCK-8 kit. Liraglutide did not affect cell viability at the concentrations employed in this experoment. Scale bar = 50 μm. Data were reported as mean ± SEM for three independent experiments (*P < 0.05 vs control group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
subsequent studies was 10 nM, which was similar to the estimated maximal concentration for a single subcutaneous dose of 0.6 mg/day (product monograph for liraglutide, Victoza). Furthermore, we explored whether liraglutide regulated cell migration. After a 24 h incubation, liraglutide attenuated the AGE-BSAinduced migration of retinal pericytes in transwell and wound-healing assays, and exendin (9–39) (100 nM) suppressed the protective effect of liraglutide (Fig. 5A and C). The number of migrating cells and the percentage of wounded healing area in AGE-BSA + liraglutide group were significantly decreased compared with the AGE-BSA group, whereas in the AGE-BSA + liraglutide + exendin (9–39) group, the two indicators were significantly increased by 1.66- and 5.04-fold compared with the AGE-BSA + liraglutide group, respectively (Fig. 5B and D). Compareing AGE-BSA + liraglutide group with AGE-BSA group, western blots showed that liraglutide significantly reduced PI3K/Akt activation, reversed the increased levels of MMP-2 and p-FAK/FAK, and significantly upregulated the low level of vinculin induced by the AGEBSA treatment (Fig. 5E and F).
manner in both transwell (Fig. 2A) and wound-healing assays (Fig. 2C). However, a statistical significant difference was not observed until the concentration of AGE-BSA was increased to 100 μg/mL. At this concentration, AGE-BSA significantly enhanced the migration of retinal pericytes by 1.84- and 6.46-fold in the two assays compared with the control group, respectively (Fig. 2B and D). Vinculin, MMP-2, and pFAK/FAK play vital roles in cell migration. Thus, we examined whether their levels were altered during AGE-BSA-mediated migration. After a 24 h incubation, 100 μg/mL AGE-BSA significantly decreased vinculin expression by 1.62-fold, and increased the levels of MMP-2 and p-FAK/ FAK in retinal pericytes by 1.37- and 1.79-fold compared with the control group, respectively (Fig. 2E and F). 3.2. The PI3K/Akt pathway was involved in AGE-BSA-induced retinal pericyte migration AGE-BSA induced the activation of the PI3K/Akt pathway in a dosedependent manner. Treatment with 100 μg/mL AGE-BSA increased the phosphorylation of Akt 1.38-fold compared with the control group (Fig. 3A and B). Next, we explored whether PI3K/Akt pathway regulated the migration of retinal pericytes. We pretreated the cells with the PI3K inhibitor, LY294002 (10 μM) for 2 h, followed by Control-BSA (100 μg/mL) or AGE-BSA (100 μg/mL) for 24 h. As shown in the images from the transwell and wound-healing assays (Fig. 3C and E), LY294002 attenuated the AGE-BSA-induced migration of retinal pericytes and significantly decreased the number of migrating cells and the percentage of the wounded healing area compared with the AGE-BSA group (Fig. 3D and F). Western blot analyses further revealed that the activation of PI3K was significantly suppressed in AGEBSA + LY294002 group compared with the AGE-BSA group, and PI3K inhibition reversed the overexpression of MMP-2; however, it did not affect the levels of p-FAK/FAK and vinculin (Fig. 3G and H).
4. Discussion Pericyte migration is an alternative or additional mechanism underlying pericyte loss in diabetic retinopathy [30], which plays a crucial role in the early impairment of the BRB and the increase in vessel permeability. In the pathogenesis of DR, the loss of pericytes occurs earlier than the formation of new capillaries. However, previous studies of vascular cell migration have primarily focused on endothelial cells and angiogenesis. A large number of AGE-BSA could induce cell apoptosis, and hence, we selected the AGE-BSA concentrations that did not decrease cell viability to examine the migration of retinal pericytes. The intervention was applied for 24 h, and thus did not produce longterm damage. Based on the results from the current study, AGE-BSA increased the migration of retinal pericytes without influencing cell viability, which might be a self-protective behavior to avoid further injuries to the cell. However, the migration of pericytes contributes to increased microvascular permeability and promotes the progression of DR. At the ultrastructural level, the separation of brain pericytes from brain microvessels has been observed at as early as 2 h after the onset of hypoxia [13]. Pericyte migration from the basement membrane has also been observed after 1 h of irreversible ischemia [10]. Thus, we speculated that retinal pericyte migration might be an early stress response to external noxious stimuli. Pericytes migrate from vessels into the perivascular parenchyma, disrupt the integrity of BRB, and further aggravate the vascular injuries. Migration is a highly integrated process that required sophisticated
3.3. Liraglutide attenuated AGE-BSA-induced retinal pericytes migration and PI3K activation We detected the expression of Glp1r in retinal pericytes using immunofluorescence staining (green, Fig. 4A). In addition, the western blot assay also revealed the expression of Glp1r in retinal pericytes; rat insulinoma cells (RIN-m5F cells) were used as the positive control (Fig. 4B). Next, cells were cultured with different concentrations of liraglutide (1, 10, 25, 50, or 100 nM) for 24 h to evaluate the cellular toxicity. Treatments with up to 100 nM liraglutide were tolerated by retinal pericytes, without a considerable effect on cell survival (Fig. 4C). Additionally, the concentration of liraglutide utilized in 11
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Fig 5. Liraglutide attenuated the AGE-BSA-induced migration of retinal pericytes. (A–D) The results of the transwell (scale bar = 200 μm) and wound-healing (10× objective lens) assays showed that liraglutide attenuated the AGE-BSA-induced migration of retinal pericytes, and exendin (9–39) suppressed the effect of liraglutide. (E–F) Liraglutide reversed the AGE-BSA-induced changes in the ratio of p-Akt/Akt levels, decreased the MMP-2 and p-FAK/FAK levels, and increased the low vinculin levels. Data were reported as mean ± SEM for three independent experiments. *P < 0.05 vs control group, **P < 0.01 vs control group, ***P < 0.001 vs control group; #P < 0.05 vs AGE-BSA group, ##P < 0.01 vs AGE-BSA group, ###P < 0.001 vs AGE-BSA group; ***P < 0.001 vs AGE-BSA + liraglutide group; P was calculated using Student’s t-test; A.U., arbitrary units.
expression decreased following treatment with AGE-BSA. MMP-2 degrades ECM components, thereby facilitating cell migration. For instance, the cleavage of laminin-5 by MMP-2 reveals a cryptic site that enhances endothelial cell migration [11]. Here, AGE-BSA simultaneously increased the migration of retinal pericytes and the expression of intracellular MMP-2, further confirming that MMP-2 also participates in promoting cell motility. In the present study, we detected the expression of Glp1r in retinal pericytes and proved that liraglutide attenuated the AGE-BSA-induced migration of retinal pericytes. GLP-1 has been reported to regulate the migration of several types of cells through the PI3K/Akt signaling pathway [38,42]. Based on the current data, the PI3K inhibitor LY294002 significantly attenuated the AGE-BSA-induced migration of retinal pericytes, and this effect was associated with the inhibition of MMP-2. The pTyr-397 site on FAK modulates the SH2 domains in several cellular signaling proteins, including PI3K [5]. Here, we found that PI3K inhibition did not influence FAK phosphorylation, thereby indicating that PI3K may be activated downstream of FAK.
regulation of structural networks and signaling throughout the cell. Among these, the leading networks include integrin-based FAs, through which the cells engage in adhesive contacts with the surrounding ECM [19]. FAK has emerged as a key signaling component at FAs since its discovery [41]. Over the past 20 years, the regulation of cell migration by integrin signaling through FAK has been well-established in many cell types [25,12]. In the active state, the amino-terminal FERM domain of FAK is displaced by an activating protein [34]. FAK then undergoes autophosphorylation at Tyr-397, in turn recruiting the Src family PTKs and resulting in the phosphorylation of Tyr-576 and Tyr-577 in the FAK activation loop and full FAK activation of the catalytic domain. In the present study, we detected the extent of FAK phosphorylation in pericytes treated with AGE-BSA and observed increased phosphorylation at Try-397, consistent with the elevated migration rate. Vinculin binds to talin and actin as an FA adaptor protein [18]. In response to the increased load, vinculin is recruited to FAs [33], and the enhanced talinvinculin-actin linkages are proposed as critical features required to strengthen adhesions [7]. The current data showed that vinculin
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Pretreatment with liraglutide attenuated the AGE-BSA-induced migration of retinal pericytes, and this effect was attenuated by the GLP-1 receptor antagonist, indicating that liraglutide exerts its protective effect via Glp1r. Thus, this phenomenon may be associated with the simultaneous inhibition of FAK phosphorylation and the suppression of PI3K/Akt activation.
[16]
[17]
5. Conclusion [18]
In summary, we postulate that retinal pericyte migration might be a self-protective behavior to avoid further cell injuries. However, the migration may contribute to increased microvascular permeability and promote the progression of DR. Moreover, we also observed the expression of Glp1r in retinal pericytes and confirmed the protective effect of liraglutide on attenuating of AGE-BSA-induced pericyte migration through Glp1r. We also confirmed that the PI3K/Akt pathway is implicated in this process. Taken together, our results provided novel insights into the mechanism underlying retinal pericyte migration, and the early use of liraglutide may modulate the migration of these cells to maintain the integrity of the BRB.
[19]
[20] [21]
[22]
[23] [24]
Conflicts of interest [25]
The authors declare no conflict of interest. [26]
Acknowledgment
[27]
This work was supported by the National Natural Science Foundation of China (81670739).
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