- Email: [email protected]
2, FAK-1, and paxillin in vitro and in vivo
Biochemical Pharmacology 93 (2015) 496–505
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Myricetin inhibits advanced glycation end product (AGE)-induced migration of retinal pericytes through phosphorylation of ERK1/2, FAK-1, and paxillin in vitro and in vivo Young Sook Kim 1, Junghyun Kim 1, Ki Mo Kim, Dong Ho Jung, Sojin Choi, Chan-Sik Kim, Jin Sook Kim * Korean Medicine-Based Herbal Drug Development Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM), 1672 Yuseongdae-ro, Yuseong-gu, Daejeon, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 August 2014 Accepted 30 September 2014 Available online 22 October 2014
Advanced glycation end products (AGE) have been implicated in the development of diabetic retinopathy. Characterization of the early stages of diabetic retinopathy is retinal pericytes loss, which is the result of pericytes migration. In this study, we investigated the pathological mechanisms of AGE on the migration of retinal pericytes and confirmed the inhibitory effect of myricetin on migration in vitro and in vivo. Migration assays of bovine retinal pericytes (BRP) were induced using AGE-BSA and phosphorylation of Src, ERK1/2, focal adhesion kinase (FAK-1) and paxillin were determined using immunoblot analysis. Sprague-Dawley rats (6 weeks old) were injected intravitreally with AGE-BSA and morphological and immunohistochemical analysis of p-FAK-1 and p-paxillin were performed in the rat retina. Immunoblot analysis and siRNA transfection were used to study the molecular mechanism of myricetin on BRP migration. AGE-BSA increased BRP migration in a dose-dependent manner via receptor for AGEs (RAGE)-dependent activation of the Src kinase-ERK1/2 pathway. AGE-BSA-induced migration was inhibited by an ERK1/2 specific inhibitor (PD98059), but not by p38 and Jun N-terminal kinase inhibitors. AGE-BSA increased FAK-1 and paxillin phosphorylation in a dose- and time-dependent manner. These increases were attenuated by PD98059 and ERK1/2 siRNA. Phosphorylation of FAK-1 and paxillin was increased in response to AGE-BSA-induced migration of rat retinal pericytes. Myricetin strongly inhibited ERK1/2 phosphorylation and significantly suppressed pericytes migration in AGEBSA-injected rats. Our results demonstrate that AGE-BSA participated in the pathophysiology of retinal pericytes migration likely through the RAGE-Src-ERK1/2-FAK-1-paxillin signaling pathway. Furthermore, myricetin suppressed phosphorylation of ERK 1/2-FAK-1-paxillin and inhibited pericytes migration. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Retinal pericytes Migration Focal adhesion kinase-1 Paxillin Myricetin
1. Introduction Advanced glycation end products (AGE) are generated from early glycation products, such as Schiff’s bases, or their derivative Amadori products in which amino acids in proteins undergo a nonenzymatic reaction with glucose and other reducing sugars [1]. The rate of AGE accumulation is increased in diabetes mellitus [2,3]. Increased AGE act on various cells including endothelial cells, pericytes, and mesangial cells, through receptors for AGE (RAGE); and they lead to chronic cellular activation with cytokine
* Corresponding author. E-mail addresses: [email protected] (Y.S. Kim), [email protected] (J.S. Kim). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bcp.2014.09.022 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
production and tissue damage [4–6]. This cellular activation is associated with increased expression of extracellular matrix proteins, vascular adhesion molecules, cytokines and growth factors, and is associated with chemotaxis, angiogenesis, oxidative stress, cell proliferation and/or programed cell death depending on the cell type and concurrent signaling [7]. Diabetic retinopathy is one of the most frequent complications of diabetes and is the leading cause of vision loss in adults under 40 years from developed countries [8]. Early diabetic retinopathy is characterized by increased vascular permeability, microaneurysm formation and loss of retinal pericytes [9]. Retinal pericytes have contractile functions, provide vascular stability [10] and accumulate AGE during diabetes. AGE not only induce growth retardation and apoptotic cell death, but they also exert an immediate toxicity to retinal pericytes in vitro [11,12]. The elevated AGE levels in the
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
retinal vessels of diabetic patients are correlated with serum AGE levels and with the severity of retinopathy by inducing a blood– retinal barrier dysfunction [13]. Pericyte migration is a key cellular feature of a wide variety of physiological processes, including the repair of blood vessel endothelial damage [9]. Cell motility requires cytoskeletal reorganization and involves cytoskeleton-associated tyrosine kinase phosphorylation and the formation of focal adhesion kinase (FAK-1) complexes [14]. FAK-1 undergoes autophosphorylation on a single tyrosine residue, which creates a binding site for SH2-containing proteins. Inhibiting FAK-1 activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK-1 deficient mice also show reduced migration [15,16]. Paxillin is a focal adhesion protein that is also phosphorylated on a tyrosine residue by a number of stimuli [17]. Paxillin interacts with FAK-1 at two sites in the amino and carboxyl termini, which are not required for targeting paxillin to focal adhesions [18]. Most of the studies concerning the effects of AGE-BSA on blood vessels have been conducted on vascular endothelial cells and smooth muscle cells [19,20]. However, the mechanisms underlying AGE-BSA influence on retinal pericyte migration are not well known. Myricetin (3,5,7,30 ,40 ,50 -hexahydroxyflavone, Cannabiscetin) is a primitive flavonoid from the Chrysobalanaceae family and found in most berries, vegetables, and various medicinal herbs [21]. Several studies suggested that myricetin protects tert-butylhydroperoxide (t-BHP)-induced oxidative stress in erythrocytes from type 2 diabetic patients and restores the renal activity of glutathione peroxidase (GPx) and xanthine oxidase (XO) in diabetic rats [22,23]. Myricetin along with other flavonoids decreases low density lipoprotein (LDL) glycation and electrophoretic mobility, which reduce the atherosclerotic risk of patients with diabetes mellitus [24]. Furthermore, myricetin has also been shown to inhibit aldose reductase (AR) and AGE, which accelerate the progression of diabetic complications [24,25]. In the present study, we investigated whether AGE-BSA induced phosphorylation of FAK-1 and its substrate paxillin and whether this signal would mediate pericyte migration in vivo and in vitro. In addition, we examined whether myricetin inhibited AGE-BSAinduced retinal pericyte migration. 2. Material and methods 2.1. Materials DMEM was purchased from WelGENE Inc. (Daegu, Korea). Collagenase (#10269638001) and dispase (#11097113001) were obtained from Roche Diagnostics (Mannheim, Germany). The asmooth muscle actin (a-SMA; sc-130616) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Desmin (ab8592) antibodies were purchased from Abcam (Cambridge, England), and PD98059, SB203580, SP600125 and PP2 were obtained from Calbiochem (San Diego, USA). Phospho-Src (#2105), phospho-FAK-1 (#3281), phospho-paxillin (#2541), phospho-ERK1/2 (#9101), phospho-38 (#9215), phospho-Jun Nterminal kinase (JNK; #9251), Src (#2109), FAK-1 (#3285), paxillin (#2542), ERK1/2 (#9102), p-38 (#9212) and JNK (#9258) antibodies were acquired from Cell Signaling Technology (Beverly, USA). All other reagents used, including bovine serum albumin (BSA; fraction V) and myricetin (#M6760), were purchased from Sigma-Aldrich (St. Louis, USA). 2.2. Cell preparation and culture Primary bovine retinal pericytes (BRP) were obtained from bovine retinal microvessels as described previously [27]. Briefly, fresh bovine retinas were dissected and homogenized in PBS
497
(pH 7.4). Retinas were minced into small pieces in PBS buffer. The homogenates were then digested at 37 8C for 30 min in a solution of 0.1% collagenase and dispase. After digestion, microvessel fragments were filtered sequentially through 70-mm and 40-mm filters. BRP were collected by centrifugation and cultured with conditioned DMEM medium containing 2 mM glutamine, 1% penicillin/streptomycin, and 20% FBS. BRP from one or two pooled primary cultures were trypsinized and replated (passage 1) onto a culture dish. BRP were characterized for homogeneity by positive staining for both a-SMA and desmin antibodies. 2.3. Preparation of AGE-BSA We prepared AGE-BSA in vitro by incubating 10 mg/ml BSA with 25 mM D-glucose in PBS buffer (pH 7.4) for 4 weeks under sterile conditions at 37 8C. Control non-glycated BSA (10 mg/ml) was prepared in the same manner but without D-glucose supplementation. AGE formation was characterized using fluorescence spectroscopy (ex 350 nm/em 450 nm). We noted a 7.5-fold increase in fluorescence when AGE-BSA was compared to control BSA. AGE-BSA was purified by PD-10 columns (Bio-Rad, Hercules, USA) and tested for endotoxin using the chromogenic LAL endotoxin assay kit (GenScript, Piscataway, USA). AGE-BSA samples contained less than 0.005 U/ml endotoxin. 2.4. Cell migration assay Migration assays were performed as previously described [14]. Briefly, the chemotactic motility of BRP was assayed using Transwell plates with 6.5-mm diameter polycarbonate filters (8-mm pore size). The lower surface of the filter was coated with gelatine (10 mg/ml). Fresh DMEM media containing AGE-BSA was placed in the lower wells. BRP were trypsinized and suspended at a final concentration of 1 106 cells/ml in DMEM containing 1% FBS. One hundred microliters of the cell suspension were loaded into each of the upper wells. Cells were fixed and stained with hematoxylin and eosin. 2.5. Immunoblot analysis Immunoblot analysis was performed as previously described [28]. Protein expression levels were determined by analyzing the signals captured on the polyvinylidene fluoride membranes using an image analyzer (Las-3000, Fuji photo, Tokyo, Japan). 2.6. Animals Adult male Sprague-Dawley (SD) rats (6 weeks old) were purchased from Koatech (Pyeongtaek, Korea) and acclimated for 1 week prior to the study. Rats were fed standard laboratory chow and allowed free access to water in an air-conditioned room with a 12-h light/12-h dark cycle until they were used for the experiment. All experimental protocols for animal care were approved by local ethical boards and animal husbandry and procedures were carried out according to institutional guidelines. 2.7. Intravitreal injection of AGE-BSA SD rats were randomly divided into three groups: normal control, AGE-BSA-treated, and AGE-BSA/myricetin-treated, with eight animals in each group. Each rat was anesthetized with a 1:1 mixture of xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (10 mg/kg). Rats received an intravitreal injection of 3 ml sterile PBS containing 1 mg/ml AGE-BSA in one eye and 5 ml sterile PBS in the contralateral eye using a 33-gauge Hamilton needle and syringe. The rats in the AGE-BSA/myricetin group
498
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
received a combination of myricetin (50 or 100 mM) and 1 mg/ml AGE-BSA by intravitreal injection. Assuming that the vitreous volume of an adult rat eye is approximately 50 ml [29], we chose the final intravitreal concentration to be 20 mg/ml AGE-BSA and myricetin (5 or 10 mM). The needle was left in position for 30–60 s and then slowly withdrawn to minimize fluid loss from the eye. Rats were monitored regularly for infection associated with the injection site. Eyes with injection-damaged lenses or retinas were
excluded from the study. Rats were anesthetized and killed 3 d after the intravitreal injection. 2.8. Trypsin digested vessel preparation Eyes were enucleated from the animals and retinas were isolated. The retinal samples were then placed in 10% formalin for 2 d. After fixation, the vessel structures were isolated from the
Fig. 1. AGE-BSA induced primary BRP migration via the pERK1/2 pathway. (A) Characterization of primary BRP (green, desmin). (B) AGE-BSA induced BRP migration. (C) Quantification of migrated BRP. Data are mean SD of three independent experiments (*P < 0.05 vs. control, **P < 0.01 vs. control). (D, E) AGE-BSA increased ERK1/2 phosphorylation in a dose- (D) and time-dependent manner (E). (F, G) AGE-BSA-stimulated BRP migration was attenuated after pretreatment with PD98059. Data are mean SD of three independent experiments (**P < 0.01 vs. control, **P < 0.01 vs. AGE-BSA-treated).
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
retinal cells by gentle rinsing in distilled water. After the vascular specimens were mounted on a slide, periodic acid-Schiff staining was performed. The specimens were then analyzed by microscope using digital capture (BX41 microscope, Olympus, Japan). The number of migrating pericytes per 0.32 mm2 of capillary area was determined by counting 10 randomly selected microscopic fields.
499
antibody. The second staining sequence using rabbit anti-paxillin (1:200) and rabbit anti-FAK (1:200) antibodies were performed on the same section with a Texas red-conjugated anti-rabbit antibody. To prevent cross-reactivity between both immunofluorescence sequences the slides were incubated with normal mouse serum after the first staining. 2.10. Statistical analysis
2.9. Double immunofluorescence staining The eyes were sectioned sagittally (4 mm), and double immunofluorescence staining was performed on the retinal sections. The first immunostain using a mouse anti-a-SMA (1:200) antibody was detected with a FITC-conjugated anti-mouse
All experiments were repeated at least three times and all values are represented as mean standard deviation. Analysis of variance was assessed using the Tukey test to determine differences (PRISM software, Graph Pad, San Diego, USA). Value of *P < 0.05 was considered statistically significant.
Fig. 2. AGE-BSA induced RAGE-dependent phosphorylation of Src and ERK1/2 in BRP. (A, B) AGE-BSA-stimulated BRP migration was attenuated by neutralizing RAGE antibodies. Data are mean SD of three independent experiments (**P < 0.01 vs. BSA, *P < 0.05 vs. AGE-BSA). (C, D) Effect of AGE-BSA on RAGE-dependent Src-ERK1/2 activation. BRPs were transfected with control siRNA or RAGE siRNA. (D) Quantification of p-Src. Data are mean SD of three independent experiments (**P < 0.01). (E, F) BRPs were pretreated with the Src inhibitor PP2 (1 mM) for 30 min and incubated in the absence or presence of AGE-BSA (100 mg/ml). (F) Quantification of p-Src. Data are mean SD of three independent experiments (**P < 0.01).
500
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
Fig. 3. AGE-BSA induced FAK and paxillin phosphorylation in BRP via ERK1/2 activation BRP were treated with different doses of AGE-BSA (A, B) or with AGE-BSA for different times (C, D). Expressions of p-FAK-1 (B) and p-paxillin (D). Data are mean SD of three independent experiments (**P < 0.01 vs. control). (E) BRP were pretreated with PD98059 or SB203580 for 30 min, and then exposed to AGE-BSA (100 mg/ml) or BSA (100 mg/ml). (F) BRPs were transfected with specific siRNA to silence ERK1/2, and exposed to AGE-BSA or BSA. (G) Levels of p-ERK. Data are mean SD from three independent experiments (**P < 0.01).
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
3. Results 3.1. AGE-BSA induced bovine retinal pericytes (BRP) migration via pERK1/2 pathway The accumulation of toxic intracellular products such as AGE influences pathogenic mechanisms in retinal cells in vitro and in vivo [30]. Migration leads to pericyte loss in diabetic retinopathy [31]. To study the effect of AGE-BSA on pericytes migration, we incubated the cells with various concentrations (25, 50, and 100 mg/ml) of AGE-BSA for 6 h. We first examined that primary BRP were characterized for homogeneity by immunoreactivity with an anti-desmin antibody, a specific pericyte marker (Fig. 1A). Increasing concentrations of AGE-BSA induced BRP migration in a dose-dependent manner (Fig. 1B and C). We next examined whether signaling by MAPK kinase family proteins (ERK1/2, p38, and JNK) is involved in AGE-BSA-stimulated pericyte migration. BRP were stimulated with various concentrations of AGE-BSA (20, 40, 80, and 100 mg/ml) for 1 h. AGE-BSA treatment enhanced ERK1/2 phosphorylation, with a maximum level of phosphorylation reached after 1 h of treatment. Furthermore, AGE-BSA increased ERK1/2 phosphorylation in a dose- and time-dependent manner, but phosphorylation of p38 and JNK did not change (Fig. 1D and E). We next performed a migration assay, which showed that AGE-BSA could significantly promote BRP migration. BRP were pretreated with ERK1/2 inhibitor PD98059 (10 mmol/l), p38 MAPK inhibitor SB203580 (10 mmol/l), or JNK inhibitor SP600125 (10 mmol/l) for 30 min, then exposed to AGE-BSA (100 mg/ml) for 6 h. AGE-BSA-stimulated BRP migration was significantly attenuated (**P < 0.01) after pretreatment with PD98059 compared to AGE-BSA alone, but neither SB203580 nor SP600125 affected migration (Fig. 1F and G).
501
3.2. AGE-BSA induced RAGE-dependent phosphorylation of Src and ERK1/2 in BRP Previous studies have reported that engagement of RAGE by AGE activates key signal transduction pathways, such as pSrc, ERK1/2 kinase, and nuclear factor-kappa B (NF-kB), in endothelial cells and vascular smooth muscle cells [32–34]. Additionally, we examined whether the AGE-BSA-induced migration is dependent on RAGE signaling. BRP were treated with BSA (100 mg/ml), or pretreated with neutralizing RAGE antibodies (100 mg/ml) and then exposed to AGE-BSA for 6 h. AGE-BSA, but not BSA, caused a 1.5-fold increase in BRP migration (Fig. 2B and C, **P < 0.01) compared to control, and BRP migration was significantly attenuated by neutralizing RAGE antibodies (Fig. 2A and B, *P < 0.05). We used the RAGE siRNA method to examine whether RAGEdependent downstream signaling pathways involved ERK1/2, p38, and JNK via Src phosphorylation. BRP transfected with control siRNA and treated with AGE-BSA had 2.0-fold higher Src phosphorylation (**P < 0.01) compared to AGE-BSA-treated RAGE siRNA-transfected cells (Fig. 2C and D). Furthermore, PP2, a specific inhibitor of Src, reduced AGE-BSA-induced Src and ERK1/2 phosphorylation (Fig. 2E and F). This suggests that AGE-BSA induces RAGE-dependent activation of Src/ERK 1/2 module in pericytes. 3.3. AGE-BSA induced FAK and paxillin phosphorylation in BRP via ERK1/2 phosphorylation The cytoskeletal proteins FAK and paxillin mediate migration in various normal and cancer cells [35–37]. In particular, the phosphorylated FAK forms a complex with the Src family of tyrosine kinases, and FAK and paxillin interact with intracellular
Fig. 4. Effect of intravitreal injection of AGE-BSA on pericytes migration in SD rats in each group. (A) Representative photomicrographs of a retinal digest preparation from a rat injected with PBS (control, n = 8) and from a rat injected with AGE-BSA (a single dose of 20 mg/eye, n = 8). The arrow indicates a migrating pericyte on a straight capillary with decreased endothelium contact (PAS, magnification 400). (B) Retinas were evaluated by quantitative retinal morphometry 2 d after injection (*P < 0.05 vs. control).
502
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
signal transduction pathways such as pERK1/2, PI3K and Akt [38]. We next examined whether the FAK-1 and paxillin are activated in response to AGE-BSA in BRP and which signaling pathways were involved. BRP were treated with various concentrations of AGE-BSA for 1 h before immunoblot analysis. FAK-1 and paxillin phosphorylation increased in a dose- and time-dependent manner (Fig. 3B and D). Pretreatment with SB203580, a p38 specific inhibitor, did not affect AGE-BSA-induced FAK-1 and paxillin phosphorylation in BRP. However, PD98059, a specific ERK1/2 inhibitor, suppressed AGE-BSA-induced ERK1/2, FAK-1 and paxillin phosphorylation in BRP (Fig. 3E). We further examined changes in FAK-1 and paxillin expression following transfection with ERK1/2 siRNA. BRP transfected with control siRNA and treated with AGE-BSA (but not BSA) increased FAK-1, paxillin, and ERK1/2 phosphorylation. However, silencing ERK1/2 with a specific siRNA abolished phosphorylation of FAK-1 and paxillin (Fig. 3F and G **P < 0.01). These data suggest that AGE-BSA stimulation induced FAK-1 and paxillin phosphorylation in pericytes by mechanisms that require ERK1/2 phosphorylation. 3.4. Intravitreal injection of AGE-BSA induced migration in rat retinal pericytes (RRP) Although hyperglycemia increases the pericytes migration [31], whether pericytes migration can increase by AGE-BSA in vivo is still uncertain. AGE-BSA was injected intravitreally into SD rats. Retinas were isolated after retinal digest preparation
and morphometry. Pericytes on straight capillaries showed small, round, or elongated nuclei. AGE-BSA-injected rats had migrating pericytes (Fig. 4A, arrow) on straight capillaries with less contact with the endothelium than the normal control (number of acellular capillaries/mm2 of retinal area, 13.75 3.37 in AGE-BSA group vs. 5.250 1.65 in control group, * P < 0.05, Fig. 4B). 3.5. Intravitreal injection of AGE-BSA increased on FAK-1 and paxillin phosphorylation in RRP As shown in Fig. 3, AGE-BSA-induced phosphorylation of FAK-1 and paxillin suggest the possibility that a variety of physiological processes including pericytes migration might be mediated and influenced. To evaluate the importance of FAK-1 and paxillin in AGE-BSA-mediated signaling in vivo, we used immunofluorescence staining to examine whether the intravitreal injection of AGE-BSA induced FAK-1 and paxillin phosphorylation. First, we used immunostaining with antibodies against a-SMA, a pericyte marker to identify retinal pericytes. Specific a-SMA (green) expression was seen in the outer capillary vessel, and intravitreal injection of AGEBSA significantly increased FAK-1 (Fig. 5A, red, arrow) and paxillin (Fig. 5D, red, arrow) phosphorylation after 2 d. Immunoblot analysis further confirmed that AGE-BSA induced FAK-1 and paxillin phosphorylation (Fig. 5B and E), FAK-1 phosphorylation increased by 1.17-fold (**P < 0.01) and paxillin phosphorylation increased by 1.23-fold (**P < 0.01) compared to the retina of normal control rats (Fig. 5C and F). These results suggest that FAK-1
Fig. 5. Effect of intravitreal injection of AGE-BSA on FAK-1 and paxillin expression in RRPs (A) Double immunofluorescence staining for a-SMA (green) and p-FAK-1(red) immunostaining (n = 8 in each group). (B, C) Immunoblot analysis of p-FAK. Data are mean SD from three independent experiments (**P < 0.01 vs. control). (D) Double immunofluorescence staining for a-SMA (green) and p-paxillin (red) (n = 8 in each group). (E, F) Immunoblot analysis of p-paxillin. Data are mean SD of three independent experiments (**P < 0.01 vs. control).
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
503
Fig. 6. Myricetin induced AGE-BSA-induced phosphorylation of ERK1/2 and cell migration. (A) Structure of myricetin. (B) BRP were pretreated with myricetin (10 mM) or PD98059 (10 mM) and exposed to AGE-BSA (100 mg/ml). (C) Expressions of p-ERK. Data are mean SD of three independent experiments (**P < 0.01 vs. control, #P < 0.01 vs. AGE-BSA). (D, E) Quantifications of migrated BRP by myricetin or PD98059. Data are mean SD of three independent experiments (**P < 0.01 vs. AGE-BSA, #P < 0.01 vs. AGE-BSA). (F) Representative photomicrographs (PAS, magnification 400). (G) Migrating pericytes in retinas (*P < 0.05 vs. control, # P < 0.05 vs. AGE-BSA-injected).
and paxillin play a role in AGE-BSA-mediated signaling by facilitating pericyte migration. 3.6. Myricetin induced AGE-BSA-induced phosphorylation of ERK1/2 and cell migration Myricetin (Fig. 6A), a flavonoid commonly found in edible berries, significantly reduces cancer cell migration [22,39]. We investigated the ability of myricetin or PD98059 to modulate the phosphorylation of the MEK-1 downstream signaling proteins ERK1/2, FAK-1, and paxillin. Myricetin (10 mM) blocked AGE-BSAinduced ERK1/2 phosphorylation similar to PD98059 (Fig. 6B and C). BRP migration was also significantly attenuated by 1.8-fold (**P < 0.01) upon pretreatment with myricetin compared to treatment with AGE-BSA alone (Fig. 6D and E). Furthermore, myricetin inhibited AGE-BSA-induced RRP migration in a dosedependent manner in vivo (Fig. 6F and G). Overall, our results suggest that myricetin inhibits retinal pericytes migration via ERK1/2, FAK-1, and paxillin pathways in vitro and in vivo. 4. Discussion Our results showed that AGE-BSA promoted migration in retinal pericytes via ERK1/2, FAK and paxillin phosphorylation. Furthermore, the phosphorylation of FAK-1 and paxillin increased with intravitreally injected AGE-BSA in rat eyes. Myricetin also
inhibited AGE-BSA-induced migration of retinal pericytes via ERK1/2, FAK-1 and paxillin phosphorylation in vitro and in vivo. Previous studies on the AGE expression in the retina focus primarily on the effect of AGE on endothelial cells, monocytes, and smooth muscle cells [20,32], whereas the signaling mechanism of AGE and retinal pericytes migration has not been investigated. We isolated pericytes from fresh bovine retinas using previously described methods [27,31]. The pericytes were identified based on their morphology, slow growth, and positive immunoreactivity to a-SMA and desmin, the latter of which is considered a relatively specific pericyte marker. Here we showed that migration was induced by intravitreal AGE-BSA injection in a subset of rat pericytes located on straight capillaries (Fig. 4). Studies on pericyte migration have been done in experimental diabetic retinopathy and pathogenesis [9,31]. FAK-1 and paxillin play a key role in cell migration [15,16,37,41]. FAK-1 is a non-receptor tyrosine kinase protein that localizes at focal adhesions or contacts [41] and is also phosphorylated at multiple serine residues (Ser910) in response to physiological stimuli [42]. Therefore, FAK-1 and paxillin may mediate signaling from the substratum via ERK1/2 to regulate pericyte migration. AGE-BSA induced FAK-1 and paxillin phosphorylation in vitro and in vivo in our study. Furthermore, in vitro AGE-BSA stimulated BRP migration in a dose-dependent manner. AGE-BSA (but not BSA) enhanced the migration of primary retinal pericytes, and this effect was abolished by a RAGE-neutralizing antibody. AGE likely influences cellular function via interaction
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
504
with RAGE [40,43]. RAGE expression is increased in both endothelium and vascular smooth muscle cells from diabetic patients and during up-regulation of vascular endothelial growth factor secretion in retinal pigment epithelial cells [34,40,44]. RAGE engagement in several cell types, including monocytes and endothelial cells, activates several downstream signaling pathways ultimately leading to NF-kB activation, with several intermediates linking AGE-BSA/RAGE to NF-kB in endothelial cells [45,46]. All these changes might be linked to AGE-induced diabetic complications. Our results are consistent with the key roles of this transcription factor in mediating the impact of RAGE ligands. Many extracellular signals regulate phosphorylation-dependent activation of MAPK family members that subsequently modulate transcriptional responses, thereby altering cellular function in normal and pathological settings. AGE have been shown to activate ERK1/2 in arterial smooth muscle cells and renal tubule cells [45] and have a chemotactic effect on cells, such as vascular smooth muscle cells [34,40]. Although AGE-BSA stimulated pericyte ERK1/2 activation in a dose- and timedependent manner in our study, a role for ERK1/2 signaling pathways in pericyte migration has not been established. AGEBSA stimulated the migration of pericytes and PD98059 significantly reduced migration, but SB203580 and SP600125 did not have an effect. To evaluate the role of ERK1/2 as a signaling intermediate coupled to stimulation of FAK-1 and paxillin, we pretreated pericytes with SB203580, a p38 MAPK inhibitor, and PD98059, an ERK1/2-specific inhibitor. Our results show that treatment with PD98059 and ERK1/2 knockdown by siRNA both completely abolished FAK-1 and paxillin phosphorylation. AGE-BSA stimulated FAK-1 and paxillin phosphorylation in pericytes, suggesting that ERK1/2 lies upstream in the signaling transduction pathway and affects FAK-1 and paxillin regulation of pericyte migration. Recent studies show that myricetin stimulates glucose transport in rat adipocytes, enhances insulin-stimulated lipogenesis and increases hepatic glycogen and glucose-6-phosphate content without hepatotoxicity in diabetic rats [45,46]. Furthermore, myricetin inhibits matrix metalloproteinase-2 protein expression and enzymatic activity in colorectal carcinoma cells [45]. Although myricetin induces apoptosis in cancer cells, the precise impact and related molecular mechanism of myricetin on migration and invasion are still unclear [39]. As anticipated, myricetin strongly inhibited retinal pericyte migration in our study (Fig. 6), suggesting it inhibited migration in vitro and in vivo possibly through the ERK1/2signaling pathway. The precise molecular mechanism by which myricetin inactivates the ERK1/2 complex will help address the bioavailability, toxicity and other effects of myricetin. In conclusion, we found that AGE-BSA-induced retinal pericyte migration was activated by the Src-ERK1/2-FAK-1-paxillin signaling pathway and that myricetin inhibits retinal pericyte migration through inactivation of this same pathway. Natural extracts with high myricetin content could be developed as phytochemicals for functional food products to prevent diabetic retinopathy. Eating berries and edible plants such as cherry, cranberry and parsley may also help prevent or delay the development of diabetic retinopathy.
Conflict of interest statement None. Acknowledgments This research was supported by grants (K13040 and K14040) from the Korea Institute of Oriental Medicine (KIOM).
References [1] Brownlee M, Cerami A, Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 1988;318:1315–21. [2] Makita Z, Vlassara H, Cerami A, Bucala R. Immunochemical detection of advanced glycosylation end products in vivo. J Biol Chem 1992;267:5133–8. [3] Nakayama H, Mitsuhashi T, Kuwajima S, Aoki S, Kuroda Y, Itoh T, et al. Immunochemical detection of advanced glycation end products in lens crystallins from streptozocin-induced diabetic rat. Diabetes 1993;42:345–50. [4] Schmidt AM, Mora R, Cao R, Yan SD, Brett J, Ramakrishnan R, et al. The endothelial cell binding site for advanced glycation end products consists of a complex: an integral membrane protein and a lactoferrin-like polypeptide. J Biol Chem 1994;269:9882–8. [5] Ritthaler U, Deng Y, Zhang Y, Greten J, Abel M, Sido B, et al. Expression of receptors for advanced glycation end products in peripheral occlusive vascular disease. Am J Pathol 1995;146:688–94. [6] Tsuji H, Iehara N, Masegi T, Imura M, Ohkawa J, Arai H, et al. Ribozyme targeting of receptor for advanced glycation end products in mouse mesangial cells. Biochem Biophys Res Commun 1998;245:583–8. [7] Thornalley PJ. Cell activation by glycated proteins. AGE receptors, receptor recognition factors and functional classification of AGEs. Cell Mol Biol 1998;44:1013–23. [8] Congdon NG, Friedman DS, Lietman T. Important causes of visual impairment in the world today. J Am Med Assoc 2003;290:2057–60. [9] Hammes HP, Lin J, Renner O, Shani M, Lundqvist A, Betsholtz C, et al. Pericytes and the pathogenesis of diabetic retinopathy. Diabetes 2002;51:3107–12. [10] von Tell D, Armulik A, Betsholtz C. Pericytes and vascular stability. Exp Cell Res 2006;312:623–9. [11] Stitt AW, Li YM, Gardiner TA, Bucala R, Archer DB, Vlassara H. Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats. Am J Pathol 1997;150: 523–31. [12] Yamagishi S, Hsu CC, Taniguchi M, Harada S, Yamamoto Y, Ohsawa K, et al. Receptor-mediated toxicity to pericytes of advanced glycosylation end products: a possible mechanism of pericyte loss in diabetic microangiopathy. Biochem Biophys Res Commun 1995;213:681–7. [13] Stitt AW, Bhaduri T, McMullen CB, Gardiner TA, Archer DB. Advanced glycation end products induce blood–retinal barrier dysfunction in normoglycemic rats. Mol Cell Biol Res Commun: MCBRC 2000;3:380–8. [14] Jimenez C, Portela RA, Mellado M, Rodrı´guez-Frade JM, Collard J, Serrano A, et al. Role of the PI3K regulatory subunit in the control of actin organization and cell migration. J Cell Biol 2000;151:249–62. [15] Gilmore AP, Romer LH. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol Biol Cell 1996;7:1209–24. [16] Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, et al. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAKdeficient mice. Nature 1995;377:539–44. [17] Hildebrand JD, Schaller MD, Parsons JT. Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase. Mol Biol Cell 1995;6:637–67. [18] Brown MC, Perrotta JA, Turner CE. Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding. J Cell Biol 1996;135:1109–23. [19] Otero K, Martinez F, Beltran A, Gonza´lez D, Herrera B, Quintero G, et al. Albumin-derived advanced glycation end-products trigger the disruption of the vascular endothelial cadherin complex in cultured human and murine endothelial cells. Biochem J 2001;359:567–74. [20] Sakata N, Meng J, Takebayashi S. Effects of advanced glycation end products on the proliferation and fibronectin production of smooth muscle cells. J Atheroscler Thromb 2000;7:169–76. [21] Miean KH, Mohamed S. Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. J Agric Food Chem 2001;49:3106–12. [22] Ding Y, Li Y. Minireview: therapeutic potential of myricetin in diabetes mellitus. Food Sci Human Well 2012;1:19–25. [23] Ong KC, Khoo HE. Biological effects of myricetin. Gen Pharmacol 1997;29: 12–6. [24] Ghaffari MA, Mojab S. Influence of flavonols as in vitro on low density lipoprotein glycation. Iran Biomed J 2007;11:185–91. [25] Urios P, Grigorova-Borsos AM, Sternberg M. Flavonoids inhibit the formation of the cross-linking AGE pentosidine in collagen incubated with glucose, according to their structure. Eur J Nutri 2007;46:139–46. [27] Nayak RC, Herman IM. Bovine retinal microvascular pericytes: isolation, propagation, and identification. Methods Mol Med 2001;46:247–63. [28] Kim YS, Kim NH, Jung DH, Jang DS, Lee YM, Kim JM, et al. Genistein inhibits aldose reductase activity and high glucose-induced TGF-beta2 expression in human lens epithelial cells. Eur J Pharmacol 2008;594: 18–25. [29] Berkowitz BA, Lukaszew RA, Mullins CM, Penn JS. Impaired hyaloidal circulation function and uncoordinated ocular growth patterns in experimental retinopathy of prematurity. Invest Ophthalmol Vis Sci 1998;39:391–6. [30] Stitt AW. AGEs diabetic retinopathy. Invest Ophthalmol Vis Sci 2010;51: 4867–74.
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505 [31] Pfister F, Feng Y, vom Hagen F, Hoffmann S, Molema G, Hillebrands JL, et al. Pericyte migration: a novel mechanism of pericyte loss in experimental diabetic retinopathy. Diabetes 2008;57:2495–502. [32] Taguchi A, Blood DC, del Toro G, Hoffmann S, Molema G, Hillebrands JL, et al. Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature 2000;405:354–60. [33] Shoji T, Koyama H, Morioka T, Tanaka S, Kizu A, Motoyama K, et al. Receptor for advanced glycation end products is involved in impaired angiogenic response in diabetes. Diabetes 2006;55:2245–55. [34] Reddy MA, Li SL, Sahar S, Kim YS, Xu ZG, Lanting L, et al. Key role of Src kinase in S100B-induced activation of the receptor for advanced glycation end products in vascular smooth muscle cells. J Biol Chem 2006;281: 13685–93. [35] Zhao X, Guan JL. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Adv Drug Deliv Rev 2011;63:610–5. [36] Tumbarello DA, Brown MC, Hetey SE, Turner CE. Regulation of paxillin family members during epithelial-mesenchymal transformation: a putative role for paxillin delta. J Cell Sci 2005;118:4849–63. [37] Yu CF, Sanders MA, Basson MD. Human caco-2 motility redistributes FAK and paxillin and activates p38 MAPK in a matrix-dependent manner. Am J Physiol Gastrointest Liver Physiol 2000;278:G952–66. [38] Schaller MD. Paxillin: a focal adhesion-associated adaptor protein. Oncogene 2001;20:6459–72.
505
[39] Sun F, Zheng XY, Ye J, Wu TT, Wang JI, Chen W. Potential anticancer activity of myricetin in human t24 bladder cancer cells both in vitro and in vivo. Nutr Cancer 2012;64:599–606. [40] Ramasamy R, Yan SF, Herold K, Clynes R, Schmidt AM. Receptor for advanced glycation end products: fundamental roles in the inflammatory response: winding the way to the pathogenesis of endothelial dysfunction and atherosclerosis. Ann N Y Acad Sci 2008;1126:7–13. [41] Schaller MD. Biochemical signals and biological responses elicited by the focal adhesion kinase. Biochim Biophys Acta 2001;1540:1–21. [42] Hunger-Glaser I, Salazar EP, Sinnett-Smith J, Rozengurt E. Bombesin, lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesion kinase phosphorylation at Ser-910: requirement for ERK activation. J Biol Chem 2003;278:22631–43. [43] Schmidt AM, Yan SD, Wautier JL, Stern D. Activation of receptor for advanced glycation end products: a mechanism for chronic vascular dysfunction in diabetic vasculopathy and atherosclerosis. Circ Res 1999;84:489–97. [44] Ma W, Lee SE, Guo J, Qu W, Hudson BI, Qu W, et al. RAGE ligand upregulation of VEGF secretion in ARPE-19 cells. Invest Ophthalmol Vis Sci 2007;48:1355–61. [45] Simm A, Mu¨nch G, Seif F, Schenk O, Heidland A, Richter H, et al. Advanced glycation endproducts stimulate the MAP-kinase pathway in tubulus cell line LLC-PK1. FEBS Lett 1997;410:481–7. [46] Ong KC, Khoo HE. Effects of myricetin on glycemia and glycogen metabolism in diabetic rats. Life Sci 2000;67:1695–705.
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Myricetin inhibits advanced glycation end product (AGE)-induced migration of retinal pericytes through phosphorylation of ERK1/2, FAK-1, and paxillin in vitro and in vivo Young Sook Kim 1, Junghyun Kim 1, Ki Mo Kim, Dong Ho Jung, Sojin Choi, Chan-Sik Kim, Jin Sook Kim * Korean Medicine-Based Herbal Drug Development Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM), 1672 Yuseongdae-ro, Yuseong-gu, Daejeon, South Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 August 2014 Accepted 30 September 2014 Available online 22 October 2014
Advanced glycation end products (AGE) have been implicated in the development of diabetic retinopathy. Characterization of the early stages of diabetic retinopathy is retinal pericytes loss, which is the result of pericytes migration. In this study, we investigated the pathological mechanisms of AGE on the migration of retinal pericytes and confirmed the inhibitory effect of myricetin on migration in vitro and in vivo. Migration assays of bovine retinal pericytes (BRP) were induced using AGE-BSA and phosphorylation of Src, ERK1/2, focal adhesion kinase (FAK-1) and paxillin were determined using immunoblot analysis. Sprague-Dawley rats (6 weeks old) were injected intravitreally with AGE-BSA and morphological and immunohistochemical analysis of p-FAK-1 and p-paxillin were performed in the rat retina. Immunoblot analysis and siRNA transfection were used to study the molecular mechanism of myricetin on BRP migration. AGE-BSA increased BRP migration in a dose-dependent manner via receptor for AGEs (RAGE)-dependent activation of the Src kinase-ERK1/2 pathway. AGE-BSA-induced migration was inhibited by an ERK1/2 specific inhibitor (PD98059), but not by p38 and Jun N-terminal kinase inhibitors. AGE-BSA increased FAK-1 and paxillin phosphorylation in a dose- and time-dependent manner. These increases were attenuated by PD98059 and ERK1/2 siRNA. Phosphorylation of FAK-1 and paxillin was increased in response to AGE-BSA-induced migration of rat retinal pericytes. Myricetin strongly inhibited ERK1/2 phosphorylation and significantly suppressed pericytes migration in AGEBSA-injected rats. Our results demonstrate that AGE-BSA participated in the pathophysiology of retinal pericytes migration likely through the RAGE-Src-ERK1/2-FAK-1-paxillin signaling pathway. Furthermore, myricetin suppressed phosphorylation of ERK 1/2-FAK-1-paxillin and inhibited pericytes migration. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Retinal pericytes Migration Focal adhesion kinase-1 Paxillin Myricetin
1. Introduction Advanced glycation end products (AGE) are generated from early glycation products, such as Schiff’s bases, or their derivative Amadori products in which amino acids in proteins undergo a nonenzymatic reaction with glucose and other reducing sugars [1]. The rate of AGE accumulation is increased in diabetes mellitus [2,3]. Increased AGE act on various cells including endothelial cells, pericytes, and mesangial cells, through receptors for AGE (RAGE); and they lead to chronic cellular activation with cytokine
* Corresponding author. E-mail addresses: [email protected] (Y.S. Kim), [email protected] (J.S. Kim). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bcp.2014.09.022 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
production and tissue damage [4–6]. This cellular activation is associated with increased expression of extracellular matrix proteins, vascular adhesion molecules, cytokines and growth factors, and is associated with chemotaxis, angiogenesis, oxidative stress, cell proliferation and/or programed cell death depending on the cell type and concurrent signaling [7]. Diabetic retinopathy is one of the most frequent complications of diabetes and is the leading cause of vision loss in adults under 40 years from developed countries [8]. Early diabetic retinopathy is characterized by increased vascular permeability, microaneurysm formation and loss of retinal pericytes [9]. Retinal pericytes have contractile functions, provide vascular stability [10] and accumulate AGE during diabetes. AGE not only induce growth retardation and apoptotic cell death, but they also exert an immediate toxicity to retinal pericytes in vitro [11,12]. The elevated AGE levels in the
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
retinal vessels of diabetic patients are correlated with serum AGE levels and with the severity of retinopathy by inducing a blood– retinal barrier dysfunction [13]. Pericyte migration is a key cellular feature of a wide variety of physiological processes, including the repair of blood vessel endothelial damage [9]. Cell motility requires cytoskeletal reorganization and involves cytoskeleton-associated tyrosine kinase phosphorylation and the formation of focal adhesion kinase (FAK-1) complexes [14]. FAK-1 undergoes autophosphorylation on a single tyrosine residue, which creates a binding site for SH2-containing proteins. Inhibiting FAK-1 activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK-1 deficient mice also show reduced migration [15,16]. Paxillin is a focal adhesion protein that is also phosphorylated on a tyrosine residue by a number of stimuli [17]. Paxillin interacts with FAK-1 at two sites in the amino and carboxyl termini, which are not required for targeting paxillin to focal adhesions [18]. Most of the studies concerning the effects of AGE-BSA on blood vessels have been conducted on vascular endothelial cells and smooth muscle cells [19,20]. However, the mechanisms underlying AGE-BSA influence on retinal pericyte migration are not well known. Myricetin (3,5,7,30 ,40 ,50 -hexahydroxyflavone, Cannabiscetin) is a primitive flavonoid from the Chrysobalanaceae family and found in most berries, vegetables, and various medicinal herbs [21]. Several studies suggested that myricetin protects tert-butylhydroperoxide (t-BHP)-induced oxidative stress in erythrocytes from type 2 diabetic patients and restores the renal activity of glutathione peroxidase (GPx) and xanthine oxidase (XO) in diabetic rats [22,23]. Myricetin along with other flavonoids decreases low density lipoprotein (LDL) glycation and electrophoretic mobility, which reduce the atherosclerotic risk of patients with diabetes mellitus [24]. Furthermore, myricetin has also been shown to inhibit aldose reductase (AR) and AGE, which accelerate the progression of diabetic complications [24,25]. In the present study, we investigated whether AGE-BSA induced phosphorylation of FAK-1 and its substrate paxillin and whether this signal would mediate pericyte migration in vivo and in vitro. In addition, we examined whether myricetin inhibited AGE-BSAinduced retinal pericyte migration. 2. Material and methods 2.1. Materials DMEM was purchased from WelGENE Inc. (Daegu, Korea). Collagenase (#10269638001) and dispase (#11097113001) were obtained from Roche Diagnostics (Mannheim, Germany). The asmooth muscle actin (a-SMA; sc-130616) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Desmin (ab8592) antibodies were purchased from Abcam (Cambridge, England), and PD98059, SB203580, SP600125 and PP2 were obtained from Calbiochem (San Diego, USA). Phospho-Src (#2105), phospho-FAK-1 (#3281), phospho-paxillin (#2541), phospho-ERK1/2 (#9101), phospho-38 (#9215), phospho-Jun Nterminal kinase (JNK; #9251), Src (#2109), FAK-1 (#3285), paxillin (#2542), ERK1/2 (#9102), p-38 (#9212) and JNK (#9258) antibodies were acquired from Cell Signaling Technology (Beverly, USA). All other reagents used, including bovine serum albumin (BSA; fraction V) and myricetin (#M6760), were purchased from Sigma-Aldrich (St. Louis, USA). 2.2. Cell preparation and culture Primary bovine retinal pericytes (BRP) were obtained from bovine retinal microvessels as described previously [27]. Briefly, fresh bovine retinas were dissected and homogenized in PBS
497
(pH 7.4). Retinas were minced into small pieces in PBS buffer. The homogenates were then digested at 37 8C for 30 min in a solution of 0.1% collagenase and dispase. After digestion, microvessel fragments were filtered sequentially through 70-mm and 40-mm filters. BRP were collected by centrifugation and cultured with conditioned DMEM medium containing 2 mM glutamine, 1% penicillin/streptomycin, and 20% FBS. BRP from one or two pooled primary cultures were trypsinized and replated (passage 1) onto a culture dish. BRP were characterized for homogeneity by positive staining for both a-SMA and desmin antibodies. 2.3. Preparation of AGE-BSA We prepared AGE-BSA in vitro by incubating 10 mg/ml BSA with 25 mM D-glucose in PBS buffer (pH 7.4) for 4 weeks under sterile conditions at 37 8C. Control non-glycated BSA (10 mg/ml) was prepared in the same manner but without D-glucose supplementation. AGE formation was characterized using fluorescence spectroscopy (ex 350 nm/em 450 nm). We noted a 7.5-fold increase in fluorescence when AGE-BSA was compared to control BSA. AGE-BSA was purified by PD-10 columns (Bio-Rad, Hercules, USA) and tested for endotoxin using the chromogenic LAL endotoxin assay kit (GenScript, Piscataway, USA). AGE-BSA samples contained less than 0.005 U/ml endotoxin. 2.4. Cell migration assay Migration assays were performed as previously described [14]. Briefly, the chemotactic motility of BRP was assayed using Transwell plates with 6.5-mm diameter polycarbonate filters (8-mm pore size). The lower surface of the filter was coated with gelatine (10 mg/ml). Fresh DMEM media containing AGE-BSA was placed in the lower wells. BRP were trypsinized and suspended at a final concentration of 1 106 cells/ml in DMEM containing 1% FBS. One hundred microliters of the cell suspension were loaded into each of the upper wells. Cells were fixed and stained with hematoxylin and eosin. 2.5. Immunoblot analysis Immunoblot analysis was performed as previously described [28]. Protein expression levels were determined by analyzing the signals captured on the polyvinylidene fluoride membranes using an image analyzer (Las-3000, Fuji photo, Tokyo, Japan). 2.6. Animals Adult male Sprague-Dawley (SD) rats (6 weeks old) were purchased from Koatech (Pyeongtaek, Korea) and acclimated for 1 week prior to the study. Rats were fed standard laboratory chow and allowed free access to water in an air-conditioned room with a 12-h light/12-h dark cycle until they were used for the experiment. All experimental protocols for animal care were approved by local ethical boards and animal husbandry and procedures were carried out according to institutional guidelines. 2.7. Intravitreal injection of AGE-BSA SD rats were randomly divided into three groups: normal control, AGE-BSA-treated, and AGE-BSA/myricetin-treated, with eight animals in each group. Each rat was anesthetized with a 1:1 mixture of xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (10 mg/kg). Rats received an intravitreal injection of 3 ml sterile PBS containing 1 mg/ml AGE-BSA in one eye and 5 ml sterile PBS in the contralateral eye using a 33-gauge Hamilton needle and syringe. The rats in the AGE-BSA/myricetin group
498
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
received a combination of myricetin (50 or 100 mM) and 1 mg/ml AGE-BSA by intravitreal injection. Assuming that the vitreous volume of an adult rat eye is approximately 50 ml [29], we chose the final intravitreal concentration to be 20 mg/ml AGE-BSA and myricetin (5 or 10 mM). The needle was left in position for 30–60 s and then slowly withdrawn to minimize fluid loss from the eye. Rats were monitored regularly for infection associated with the injection site. Eyes with injection-damaged lenses or retinas were
excluded from the study. Rats were anesthetized and killed 3 d after the intravitreal injection. 2.8. Trypsin digested vessel preparation Eyes were enucleated from the animals and retinas were isolated. The retinal samples were then placed in 10% formalin for 2 d. After fixation, the vessel structures were isolated from the
Fig. 1. AGE-BSA induced primary BRP migration via the pERK1/2 pathway. (A) Characterization of primary BRP (green, desmin). (B) AGE-BSA induced BRP migration. (C) Quantification of migrated BRP. Data are mean SD of three independent experiments (*P < 0.05 vs. control, **P < 0.01 vs. control). (D, E) AGE-BSA increased ERK1/2 phosphorylation in a dose- (D) and time-dependent manner (E). (F, G) AGE-BSA-stimulated BRP migration was attenuated after pretreatment with PD98059. Data are mean SD of three independent experiments (**P < 0.01 vs. control, **P < 0.01 vs. AGE-BSA-treated).
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
retinal cells by gentle rinsing in distilled water. After the vascular specimens were mounted on a slide, periodic acid-Schiff staining was performed. The specimens were then analyzed by microscope using digital capture (BX41 microscope, Olympus, Japan). The number of migrating pericytes per 0.32 mm2 of capillary area was determined by counting 10 randomly selected microscopic fields.
499
antibody. The second staining sequence using rabbit anti-paxillin (1:200) and rabbit anti-FAK (1:200) antibodies were performed on the same section with a Texas red-conjugated anti-rabbit antibody. To prevent cross-reactivity between both immunofluorescence sequences the slides were incubated with normal mouse serum after the first staining. 2.10. Statistical analysis
2.9. Double immunofluorescence staining The eyes were sectioned sagittally (4 mm), and double immunofluorescence staining was performed on the retinal sections. The first immunostain using a mouse anti-a-SMA (1:200) antibody was detected with a FITC-conjugated anti-mouse
All experiments were repeated at least three times and all values are represented as mean standard deviation. Analysis of variance was assessed using the Tukey test to determine differences (PRISM software, Graph Pad, San Diego, USA). Value of *P < 0.05 was considered statistically significant.
Fig. 2. AGE-BSA induced RAGE-dependent phosphorylation of Src and ERK1/2 in BRP. (A, B) AGE-BSA-stimulated BRP migration was attenuated by neutralizing RAGE antibodies. Data are mean SD of three independent experiments (**P < 0.01 vs. BSA, *P < 0.05 vs. AGE-BSA). (C, D) Effect of AGE-BSA on RAGE-dependent Src-ERK1/2 activation. BRPs were transfected with control siRNA or RAGE siRNA. (D) Quantification of p-Src. Data are mean SD of three independent experiments (**P < 0.01). (E, F) BRPs were pretreated with the Src inhibitor PP2 (1 mM) for 30 min and incubated in the absence or presence of AGE-BSA (100 mg/ml). (F) Quantification of p-Src. Data are mean SD of three independent experiments (**P < 0.01).
500
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
Fig. 3. AGE-BSA induced FAK and paxillin phosphorylation in BRP via ERK1/2 activation BRP were treated with different doses of AGE-BSA (A, B) or with AGE-BSA for different times (C, D). Expressions of p-FAK-1 (B) and p-paxillin (D). Data are mean SD of three independent experiments (**P < 0.01 vs. control). (E) BRP were pretreated with PD98059 or SB203580 for 30 min, and then exposed to AGE-BSA (100 mg/ml) or BSA (100 mg/ml). (F) BRPs were transfected with specific siRNA to silence ERK1/2, and exposed to AGE-BSA or BSA. (G) Levels of p-ERK. Data are mean SD from three independent experiments (**P < 0.01).
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
3. Results 3.1. AGE-BSA induced bovine retinal pericytes (BRP) migration via pERK1/2 pathway The accumulation of toxic intracellular products such as AGE influences pathogenic mechanisms in retinal cells in vitro and in vivo [30]. Migration leads to pericyte loss in diabetic retinopathy [31]. To study the effect of AGE-BSA on pericytes migration, we incubated the cells with various concentrations (25, 50, and 100 mg/ml) of AGE-BSA for 6 h. We first examined that primary BRP were characterized for homogeneity by immunoreactivity with an anti-desmin antibody, a specific pericyte marker (Fig. 1A). Increasing concentrations of AGE-BSA induced BRP migration in a dose-dependent manner (Fig. 1B and C). We next examined whether signaling by MAPK kinase family proteins (ERK1/2, p38, and JNK) is involved in AGE-BSA-stimulated pericyte migration. BRP were stimulated with various concentrations of AGE-BSA (20, 40, 80, and 100 mg/ml) for 1 h. AGE-BSA treatment enhanced ERK1/2 phosphorylation, with a maximum level of phosphorylation reached after 1 h of treatment. Furthermore, AGE-BSA increased ERK1/2 phosphorylation in a dose- and time-dependent manner, but phosphorylation of p38 and JNK did not change (Fig. 1D and E). We next performed a migration assay, which showed that AGE-BSA could significantly promote BRP migration. BRP were pretreated with ERK1/2 inhibitor PD98059 (10 mmol/l), p38 MAPK inhibitor SB203580 (10 mmol/l), or JNK inhibitor SP600125 (10 mmol/l) for 30 min, then exposed to AGE-BSA (100 mg/ml) for 6 h. AGE-BSA-stimulated BRP migration was significantly attenuated (**P < 0.01) after pretreatment with PD98059 compared to AGE-BSA alone, but neither SB203580 nor SP600125 affected migration (Fig. 1F and G).
501
3.2. AGE-BSA induced RAGE-dependent phosphorylation of Src and ERK1/2 in BRP Previous studies have reported that engagement of RAGE by AGE activates key signal transduction pathways, such as pSrc, ERK1/2 kinase, and nuclear factor-kappa B (NF-kB), in endothelial cells and vascular smooth muscle cells [32–34]. Additionally, we examined whether the AGE-BSA-induced migration is dependent on RAGE signaling. BRP were treated with BSA (100 mg/ml), or pretreated with neutralizing RAGE antibodies (100 mg/ml) and then exposed to AGE-BSA for 6 h. AGE-BSA, but not BSA, caused a 1.5-fold increase in BRP migration (Fig. 2B and C, **P < 0.01) compared to control, and BRP migration was significantly attenuated by neutralizing RAGE antibodies (Fig. 2A and B, *P < 0.05). We used the RAGE siRNA method to examine whether RAGEdependent downstream signaling pathways involved ERK1/2, p38, and JNK via Src phosphorylation. BRP transfected with control siRNA and treated with AGE-BSA had 2.0-fold higher Src phosphorylation (**P < 0.01) compared to AGE-BSA-treated RAGE siRNA-transfected cells (Fig. 2C and D). Furthermore, PP2, a specific inhibitor of Src, reduced AGE-BSA-induced Src and ERK1/2 phosphorylation (Fig. 2E and F). This suggests that AGE-BSA induces RAGE-dependent activation of Src/ERK 1/2 module in pericytes. 3.3. AGE-BSA induced FAK and paxillin phosphorylation in BRP via ERK1/2 phosphorylation The cytoskeletal proteins FAK and paxillin mediate migration in various normal and cancer cells [35–37]. In particular, the phosphorylated FAK forms a complex with the Src family of tyrosine kinases, and FAK and paxillin interact with intracellular
Fig. 4. Effect of intravitreal injection of AGE-BSA on pericytes migration in SD rats in each group. (A) Representative photomicrographs of a retinal digest preparation from a rat injected with PBS (control, n = 8) and from a rat injected with AGE-BSA (a single dose of 20 mg/eye, n = 8). The arrow indicates a migrating pericyte on a straight capillary with decreased endothelium contact (PAS, magnification 400). (B) Retinas were evaluated by quantitative retinal morphometry 2 d after injection (*P < 0.05 vs. control).
502
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
signal transduction pathways such as pERK1/2, PI3K and Akt [38]. We next examined whether the FAK-1 and paxillin are activated in response to AGE-BSA in BRP and which signaling pathways were involved. BRP were treated with various concentrations of AGE-BSA for 1 h before immunoblot analysis. FAK-1 and paxillin phosphorylation increased in a dose- and time-dependent manner (Fig. 3B and D). Pretreatment with SB203580, a p38 specific inhibitor, did not affect AGE-BSA-induced FAK-1 and paxillin phosphorylation in BRP. However, PD98059, a specific ERK1/2 inhibitor, suppressed AGE-BSA-induced ERK1/2, FAK-1 and paxillin phosphorylation in BRP (Fig. 3E). We further examined changes in FAK-1 and paxillin expression following transfection with ERK1/2 siRNA. BRP transfected with control siRNA and treated with AGE-BSA (but not BSA) increased FAK-1, paxillin, and ERK1/2 phosphorylation. However, silencing ERK1/2 with a specific siRNA abolished phosphorylation of FAK-1 and paxillin (Fig. 3F and G **P < 0.01). These data suggest that AGE-BSA stimulation induced FAK-1 and paxillin phosphorylation in pericytes by mechanisms that require ERK1/2 phosphorylation. 3.4. Intravitreal injection of AGE-BSA induced migration in rat retinal pericytes (RRP) Although hyperglycemia increases the pericytes migration [31], whether pericytes migration can increase by AGE-BSA in vivo is still uncertain. AGE-BSA was injected intravitreally into SD rats. Retinas were isolated after retinal digest preparation
and morphometry. Pericytes on straight capillaries showed small, round, or elongated nuclei. AGE-BSA-injected rats had migrating pericytes (Fig. 4A, arrow) on straight capillaries with less contact with the endothelium than the normal control (number of acellular capillaries/mm2 of retinal area, 13.75 3.37 in AGE-BSA group vs. 5.250 1.65 in control group, * P < 0.05, Fig. 4B). 3.5. Intravitreal injection of AGE-BSA increased on FAK-1 and paxillin phosphorylation in RRP As shown in Fig. 3, AGE-BSA-induced phosphorylation of FAK-1 and paxillin suggest the possibility that a variety of physiological processes including pericytes migration might be mediated and influenced. To evaluate the importance of FAK-1 and paxillin in AGE-BSA-mediated signaling in vivo, we used immunofluorescence staining to examine whether the intravitreal injection of AGE-BSA induced FAK-1 and paxillin phosphorylation. First, we used immunostaining with antibodies against a-SMA, a pericyte marker to identify retinal pericytes. Specific a-SMA (green) expression was seen in the outer capillary vessel, and intravitreal injection of AGEBSA significantly increased FAK-1 (Fig. 5A, red, arrow) and paxillin (Fig. 5D, red, arrow) phosphorylation after 2 d. Immunoblot analysis further confirmed that AGE-BSA induced FAK-1 and paxillin phosphorylation (Fig. 5B and E), FAK-1 phosphorylation increased by 1.17-fold (**P < 0.01) and paxillin phosphorylation increased by 1.23-fold (**P < 0.01) compared to the retina of normal control rats (Fig. 5C and F). These results suggest that FAK-1
Fig. 5. Effect of intravitreal injection of AGE-BSA on FAK-1 and paxillin expression in RRPs (A) Double immunofluorescence staining for a-SMA (green) and p-FAK-1(red) immunostaining (n = 8 in each group). (B, C) Immunoblot analysis of p-FAK. Data are mean SD from three independent experiments (**P < 0.01 vs. control). (D) Double immunofluorescence staining for a-SMA (green) and p-paxillin (red) (n = 8 in each group). (E, F) Immunoblot analysis of p-paxillin. Data are mean SD of three independent experiments (**P < 0.01 vs. control).
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
503
Fig. 6. Myricetin induced AGE-BSA-induced phosphorylation of ERK1/2 and cell migration. (A) Structure of myricetin. (B) BRP were pretreated with myricetin (10 mM) or PD98059 (10 mM) and exposed to AGE-BSA (100 mg/ml). (C) Expressions of p-ERK. Data are mean SD of three independent experiments (**P < 0.01 vs. control, #P < 0.01 vs. AGE-BSA). (D, E) Quantifications of migrated BRP by myricetin or PD98059. Data are mean SD of three independent experiments (**P < 0.01 vs. AGE-BSA, #P < 0.01 vs. AGE-BSA). (F) Representative photomicrographs (PAS, magnification 400). (G) Migrating pericytes in retinas (*P < 0.05 vs. control, # P < 0.05 vs. AGE-BSA-injected).
and paxillin play a role in AGE-BSA-mediated signaling by facilitating pericyte migration. 3.6. Myricetin induced AGE-BSA-induced phosphorylation of ERK1/2 and cell migration Myricetin (Fig. 6A), a flavonoid commonly found in edible berries, significantly reduces cancer cell migration [22,39]. We investigated the ability of myricetin or PD98059 to modulate the phosphorylation of the MEK-1 downstream signaling proteins ERK1/2, FAK-1, and paxillin. Myricetin (10 mM) blocked AGE-BSAinduced ERK1/2 phosphorylation similar to PD98059 (Fig. 6B and C). BRP migration was also significantly attenuated by 1.8-fold (**P < 0.01) upon pretreatment with myricetin compared to treatment with AGE-BSA alone (Fig. 6D and E). Furthermore, myricetin inhibited AGE-BSA-induced RRP migration in a dosedependent manner in vivo (Fig. 6F and G). Overall, our results suggest that myricetin inhibits retinal pericytes migration via ERK1/2, FAK-1, and paxillin pathways in vitro and in vivo. 4. Discussion Our results showed that AGE-BSA promoted migration in retinal pericytes via ERK1/2, FAK and paxillin phosphorylation. Furthermore, the phosphorylation of FAK-1 and paxillin increased with intravitreally injected AGE-BSA in rat eyes. Myricetin also
inhibited AGE-BSA-induced migration of retinal pericytes via ERK1/2, FAK-1 and paxillin phosphorylation in vitro and in vivo. Previous studies on the AGE expression in the retina focus primarily on the effect of AGE on endothelial cells, monocytes, and smooth muscle cells [20,32], whereas the signaling mechanism of AGE and retinal pericytes migration has not been investigated. We isolated pericytes from fresh bovine retinas using previously described methods [27,31]. The pericytes were identified based on their morphology, slow growth, and positive immunoreactivity to a-SMA and desmin, the latter of which is considered a relatively specific pericyte marker. Here we showed that migration was induced by intravitreal AGE-BSA injection in a subset of rat pericytes located on straight capillaries (Fig. 4). Studies on pericyte migration have been done in experimental diabetic retinopathy and pathogenesis [9,31]. FAK-1 and paxillin play a key role in cell migration [15,16,37,41]. FAK-1 is a non-receptor tyrosine kinase protein that localizes at focal adhesions or contacts [41] and is also phosphorylated at multiple serine residues (Ser910) in response to physiological stimuli [42]. Therefore, FAK-1 and paxillin may mediate signaling from the substratum via ERK1/2 to regulate pericyte migration. AGE-BSA induced FAK-1 and paxillin phosphorylation in vitro and in vivo in our study. Furthermore, in vitro AGE-BSA stimulated BRP migration in a dose-dependent manner. AGE-BSA (but not BSA) enhanced the migration of primary retinal pericytes, and this effect was abolished by a RAGE-neutralizing antibody. AGE likely influences cellular function via interaction
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505
504
with RAGE [40,43]. RAGE expression is increased in both endothelium and vascular smooth muscle cells from diabetic patients and during up-regulation of vascular endothelial growth factor secretion in retinal pigment epithelial cells [34,40,44]. RAGE engagement in several cell types, including monocytes and endothelial cells, activates several downstream signaling pathways ultimately leading to NF-kB activation, with several intermediates linking AGE-BSA/RAGE to NF-kB in endothelial cells [45,46]. All these changes might be linked to AGE-induced diabetic complications. Our results are consistent with the key roles of this transcription factor in mediating the impact of RAGE ligands. Many extracellular signals regulate phosphorylation-dependent activation of MAPK family members that subsequently modulate transcriptional responses, thereby altering cellular function in normal and pathological settings. AGE have been shown to activate ERK1/2 in arterial smooth muscle cells and renal tubule cells [45] and have a chemotactic effect on cells, such as vascular smooth muscle cells [34,40]. Although AGE-BSA stimulated pericyte ERK1/2 activation in a dose- and timedependent manner in our study, a role for ERK1/2 signaling pathways in pericyte migration has not been established. AGEBSA stimulated the migration of pericytes and PD98059 significantly reduced migration, but SB203580 and SP600125 did not have an effect. To evaluate the role of ERK1/2 as a signaling intermediate coupled to stimulation of FAK-1 and paxillin, we pretreated pericytes with SB203580, a p38 MAPK inhibitor, and PD98059, an ERK1/2-specific inhibitor. Our results show that treatment with PD98059 and ERK1/2 knockdown by siRNA both completely abolished FAK-1 and paxillin phosphorylation. AGE-BSA stimulated FAK-1 and paxillin phosphorylation in pericytes, suggesting that ERK1/2 lies upstream in the signaling transduction pathway and affects FAK-1 and paxillin regulation of pericyte migration. Recent studies show that myricetin stimulates glucose transport in rat adipocytes, enhances insulin-stimulated lipogenesis and increases hepatic glycogen and glucose-6-phosphate content without hepatotoxicity in diabetic rats [45,46]. Furthermore, myricetin inhibits matrix metalloproteinase-2 protein expression and enzymatic activity in colorectal carcinoma cells [45]. Although myricetin induces apoptosis in cancer cells, the precise impact and related molecular mechanism of myricetin on migration and invasion are still unclear [39]. As anticipated, myricetin strongly inhibited retinal pericyte migration in our study (Fig. 6), suggesting it inhibited migration in vitro and in vivo possibly through the ERK1/2signaling pathway. The precise molecular mechanism by which myricetin inactivates the ERK1/2 complex will help address the bioavailability, toxicity and other effects of myricetin. In conclusion, we found that AGE-BSA-induced retinal pericyte migration was activated by the Src-ERK1/2-FAK-1-paxillin signaling pathway and that myricetin inhibits retinal pericyte migration through inactivation of this same pathway. Natural extracts with high myricetin content could be developed as phytochemicals for functional food products to prevent diabetic retinopathy. Eating berries and edible plants such as cherry, cranberry and parsley may also help prevent or delay the development of diabetic retinopathy.
Conflict of interest statement None. Acknowledgments This research was supported by grants (K13040 and K14040) from the Korea Institute of Oriental Medicine (KIOM).
References [1] Brownlee M, Cerami A, Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 1988;318:1315–21. [2] Makita Z, Vlassara H, Cerami A, Bucala R. Immunochemical detection of advanced glycosylation end products in vivo. J Biol Chem 1992;267:5133–8. [3] Nakayama H, Mitsuhashi T, Kuwajima S, Aoki S, Kuroda Y, Itoh T, et al. Immunochemical detection of advanced glycation end products in lens crystallins from streptozocin-induced diabetic rat. Diabetes 1993;42:345–50. [4] Schmidt AM, Mora R, Cao R, Yan SD, Brett J, Ramakrishnan R, et al. The endothelial cell binding site for advanced glycation end products consists of a complex: an integral membrane protein and a lactoferrin-like polypeptide. J Biol Chem 1994;269:9882–8. [5] Ritthaler U, Deng Y, Zhang Y, Greten J, Abel M, Sido B, et al. Expression of receptors for advanced glycation end products in peripheral occlusive vascular disease. Am J Pathol 1995;146:688–94. [6] Tsuji H, Iehara N, Masegi T, Imura M, Ohkawa J, Arai H, et al. Ribozyme targeting of receptor for advanced glycation end products in mouse mesangial cells. Biochem Biophys Res Commun 1998;245:583–8. [7] Thornalley PJ. Cell activation by glycated proteins. AGE receptors, receptor recognition factors and functional classification of AGEs. Cell Mol Biol 1998;44:1013–23. [8] Congdon NG, Friedman DS, Lietman T. Important causes of visual impairment in the world today. J Am Med Assoc 2003;290:2057–60. [9] Hammes HP, Lin J, Renner O, Shani M, Lundqvist A, Betsholtz C, et al. Pericytes and the pathogenesis of diabetic retinopathy. Diabetes 2002;51:3107–12. [10] von Tell D, Armulik A, Betsholtz C. Pericytes and vascular stability. Exp Cell Res 2006;312:623–9. [11] Stitt AW, Li YM, Gardiner TA, Bucala R, Archer DB, Vlassara H. Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats. Am J Pathol 1997;150: 523–31. [12] Yamagishi S, Hsu CC, Taniguchi M, Harada S, Yamamoto Y, Ohsawa K, et al. Receptor-mediated toxicity to pericytes of advanced glycosylation end products: a possible mechanism of pericyte loss in diabetic microangiopathy. Biochem Biophys Res Commun 1995;213:681–7. [13] Stitt AW, Bhaduri T, McMullen CB, Gardiner TA, Archer DB. Advanced glycation end products induce blood–retinal barrier dysfunction in normoglycemic rats. Mol Cell Biol Res Commun: MCBRC 2000;3:380–8. [14] Jimenez C, Portela RA, Mellado M, Rodrı´guez-Frade JM, Collard J, Serrano A, et al. Role of the PI3K regulatory subunit in the control of actin organization and cell migration. J Cell Biol 2000;151:249–62. [15] Gilmore AP, Romer LH. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol Biol Cell 1996;7:1209–24. [16] Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, et al. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAKdeficient mice. Nature 1995;377:539–44. [17] Hildebrand JD, Schaller MD, Parsons JT. Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase. Mol Biol Cell 1995;6:637–67. [18] Brown MC, Perrotta JA, Turner CE. Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding. J Cell Biol 1996;135:1109–23. [19] Otero K, Martinez F, Beltran A, Gonza´lez D, Herrera B, Quintero G, et al. Albumin-derived advanced glycation end-products trigger the disruption of the vascular endothelial cadherin complex in cultured human and murine endothelial cells. Biochem J 2001;359:567–74. [20] Sakata N, Meng J, Takebayashi S. Effects of advanced glycation end products on the proliferation and fibronectin production of smooth muscle cells. J Atheroscler Thromb 2000;7:169–76. [21] Miean KH, Mohamed S. Flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants. J Agric Food Chem 2001;49:3106–12. [22] Ding Y, Li Y. Minireview: therapeutic potential of myricetin in diabetes mellitus. Food Sci Human Well 2012;1:19–25. [23] Ong KC, Khoo HE. Biological effects of myricetin. Gen Pharmacol 1997;29: 12–6. [24] Ghaffari MA, Mojab S. Influence of flavonols as in vitro on low density lipoprotein glycation. Iran Biomed J 2007;11:185–91. [25] Urios P, Grigorova-Borsos AM, Sternberg M. Flavonoids inhibit the formation of the cross-linking AGE pentosidine in collagen incubated with glucose, according to their structure. Eur J Nutri 2007;46:139–46. [27] Nayak RC, Herman IM. Bovine retinal microvascular pericytes: isolation, propagation, and identification. Methods Mol Med 2001;46:247–63. [28] Kim YS, Kim NH, Jung DH, Jang DS, Lee YM, Kim JM, et al. Genistein inhibits aldose reductase activity and high glucose-induced TGF-beta2 expression in human lens epithelial cells. Eur J Pharmacol 2008;594: 18–25. [29] Berkowitz BA, Lukaszew RA, Mullins CM, Penn JS. Impaired hyaloidal circulation function and uncoordinated ocular growth patterns in experimental retinopathy of prematurity. Invest Ophthalmol Vis Sci 1998;39:391–6. [30] Stitt AW. AGEs diabetic retinopathy. Invest Ophthalmol Vis Sci 2010;51: 4867–74.
Y.S. Kim et al. / Biochemical Pharmacology 93 (2015) 496–505 [31] Pfister F, Feng Y, vom Hagen F, Hoffmann S, Molema G, Hillebrands JL, et al. Pericyte migration: a novel mechanism of pericyte loss in experimental diabetic retinopathy. Diabetes 2008;57:2495–502. [32] Taguchi A, Blood DC, del Toro G, Hoffmann S, Molema G, Hillebrands JL, et al. Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature 2000;405:354–60. [33] Shoji T, Koyama H, Morioka T, Tanaka S, Kizu A, Motoyama K, et al. Receptor for advanced glycation end products is involved in impaired angiogenic response in diabetes. Diabetes 2006;55:2245–55. [34] Reddy MA, Li SL, Sahar S, Kim YS, Xu ZG, Lanting L, et al. Key role of Src kinase in S100B-induced activation of the receptor for advanced glycation end products in vascular smooth muscle cells. J Biol Chem 2006;281: 13685–93. [35] Zhao X, Guan JL. Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis. Adv Drug Deliv Rev 2011;63:610–5. [36] Tumbarello DA, Brown MC, Hetey SE, Turner CE. Regulation of paxillin family members during epithelial-mesenchymal transformation: a putative role for paxillin delta. J Cell Sci 2005;118:4849–63. [37] Yu CF, Sanders MA, Basson MD. Human caco-2 motility redistributes FAK and paxillin and activates p38 MAPK in a matrix-dependent manner. Am J Physiol Gastrointest Liver Physiol 2000;278:G952–66. [38] Schaller MD. Paxillin: a focal adhesion-associated adaptor protein. Oncogene 2001;20:6459–72.
505
[39] Sun F, Zheng XY, Ye J, Wu TT, Wang JI, Chen W. Potential anticancer activity of myricetin in human t24 bladder cancer cells both in vitro and in vivo. Nutr Cancer 2012;64:599–606. [40] Ramasamy R, Yan SF, Herold K, Clynes R, Schmidt AM. Receptor for advanced glycation end products: fundamental roles in the inflammatory response: winding the way to the pathogenesis of endothelial dysfunction and atherosclerosis. Ann N Y Acad Sci 2008;1126:7–13. [41] Schaller MD. Biochemical signals and biological responses elicited by the focal adhesion kinase. Biochim Biophys Acta 2001;1540:1–21. [42] Hunger-Glaser I, Salazar EP, Sinnett-Smith J, Rozengurt E. Bombesin, lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesion kinase phosphorylation at Ser-910: requirement for ERK activation. J Biol Chem 2003;278:22631–43. [43] Schmidt AM, Yan SD, Wautier JL, Stern D. Activation of receptor for advanced glycation end products: a mechanism for chronic vascular dysfunction in diabetic vasculopathy and atherosclerosis. Circ Res 1999;84:489–97. [44] Ma W, Lee SE, Guo J, Qu W, Hudson BI, Qu W, et al. RAGE ligand upregulation of VEGF secretion in ARPE-19 cells. Invest Ophthalmol Vis Sci 2007;48:1355–61. [45] Simm A, Mu¨nch G, Seif F, Schenk O, Heidland A, Richter H, et al. Advanced glycation endproducts stimulate the MAP-kinase pathway in tubulus cell line LLC-PK1. FEBS Lett 1997;410:481–7. [46] Ong KC, Khoo HE. Effects of myricetin on glycemia and glycogen metabolism in diabetic rats. Life Sci 2000;67:1695–705.