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Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo
Biochemical and Biophysical Research Communications xxx (2018) 1e7
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Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo Kaixuan Cui b, 1, Shanshan Zhang c, 1, Xiaojuan Liu d, e, Zhenzhen Yan a, Lili Huang f, Xiaowei Yang a, Rongrong Zhu a, **, Aimin Sang a, * a
Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, 226001, China School of Medicine, Nantong University, Nantong, 226001, Jiangsu, China Nantong University, Nantong, Jiangsu, China d Department of Pathogen Biology, Medical College, Nantong University, Nantong, 226001, Jiangsu, China e Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, 226001, Jiangsu, China f Department of Ophthalmology, The First People's Hospital of Nantong, 226001, Jiangsu Province, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2018 Accepted 1 June 2018 Available online xxx
choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), causes severe vision loss among elderly patients. TANK-binding kinase 1 (TBK1) is a ubiquitously expressed serineethreonine kinase and is found to induce endothelial cells proliferation, represent a novel mediator of tumor angiogenesis and exert pro-inflammatory effect. However, the role of TBK1 in choroidal neovascularization has not been investigated so far. In this study, we found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model. Silencing of TBK1 suppressed the proliferation and tube formation activity of RF/6 A cells. Intravitreal injection of anti-TBK1 monoclonal antibody ameliorates CNV formation. Taken together, these findings exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD. © 2018 Elsevier Inc. All rights reserved.
Keywords: Choroidal neovascularization (CNV) Age-related macular degeneration (AMD) TANK-Binding kinase 1 (TBK1) Vascular endothelial growth factor (VEGF)
1. Introduction Visual impairment is a public health problem that affects patients' quality of life. Among all causes of visual impairment, agerelated macular degeneration (AMD) is a leading cause of adult blindness in industrialized countries [1]. A study forecasted that in 2020, global projected cases of any AMD would be 196 million, rising to 288 million in 2040, with the largest number of cases in Asia [2]. Early AMD is characterized by the presence of drusen, pigmentary changes at the macula, without vision loss. There are two forms of advanced AMD, which is associated with vision loss. One is atrophic form (dry AMD), which is characterized by regional retinal pigment epithelium (RPE) loss and eventual degeneration of
* Corresponding author. Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, 226001, China ** Corresponding author. Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, 226001, China E-mail addresses: [email protected] (R. Zhu), [email protected] (A. Sang). 1 These authors contributed equally to this study.
overlying photoreceptors. The other is neovascular form (wet AMD), which is characterized by choroidal neovascularization (CNV). CNV is the growth of blood vessels from the choroid through Bruch's membrane toward the retina, and it is responsible for approximately 80% of cases of severe vision loss due to AMD [3e5]. So far, the treatment of CNV remains one of the hot spots and difficulties in the field of ophthalmology. Vascular endothelial growth factor (VEGF) plays an important role in the pathophysiological process of neovascular AMD (nAMD). Anti-VEGF therapy is currently the standard of care for nAMD [6]. However, this treatment is still controversial and some patients are non-responsive or refractory to anti-VEGF therapy. Moreover, long-term anti-VEGF therapies may not prevent disease progression even in those patients who showed an initial beneficial therapeutic response, and may lead to detrimental effects on retinal function [7,8]. Therefore, we urgently need to find some molecules to regulate the expression and function of VEGF in order to treat AMD. TANK-binding kinase 1 (TBK1), also known as NF-kB-activating kinase (NAK) or T2K, is a ubiquitously expressed serineethreonine
https://doi.org/10.1016/j.bbrc.2018.06.003 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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K. Cui et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
kinase and it belongs to the non-canonical IkB kinases (IKKs) recognized for its critical role in regulating type I interferon (IFN) production, IFN regulatory factor 3 (IRF3) and NF-kB signaling pathways. TBK1 is an 84 kDa, 729-amino acid protein containing an N-terminal kinase domain, an ubiquitin like domain and two Cterminal coiled coil domains. As a non-canonical IKK, TBK1 is structurally similar to IKKε (another IKK-related kinase) and the canonical IKKs, IKKa and IKKb. It can be activated by viral infections, inflammatory mediators and so on. Previous research found that TBK1 is involved in the activation of various cellular pathways and pro-inflammatory cytokine production, and homeostatic cellular functions such as cell growth and proliferation [9e12]. Moreover, TBK1 induces human umbilical vein endothelial cells (HUVECs) proliferation, represents a novel mediator of tumor angiogenesis and exerts pro-inflammatory effects via upregulation of inflammatory cytokines. However, the role of TBK1 in CNV has not been investigated so far although several data hint at important TBK1 functions, particularly in angiogenesis and inflammation [13e15]. In the present study, we investigated the effects of TBK1 on the development of CNV, and explored the molecular mechanism. We found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model, and knockdown of TBK1 alleviates CNV formation via inhibiting VEGF expression. Our results indicate that TBK1 plays a critical role in the development of CNV and suggest TBK1 may be a potential therapeutic target for human AMD treatment. 2. Materials and methods 2.1. Animals All experimental steps were strictly performed in accordance with the requirements of the Animal Welfare Committee of Nantong University. This study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures for the use of animals were approved by the Center for Laboratory Animals of Nantong University (Nantong, Jiangsu, China). 2.2. Cell culture
with TBK1 siRNA and scramble siRNA, were placed in the wells. Medium with 200 mM CoCl2 was added and changed every 2 days. At the end of each time point, 20 ml of MTT (Sigma, St. Louis, MO) was added into each well for 4 h, then the supernatant was discarded, and 150 ml dimethyl sulfoxide (DMSO) was added for 15 min. The absorbance at 570 nm was recorded using a Microplate Reader (Model 680, Bio-Rad, Hercules, CA). The experiments were repeated at least in triplicate. 2.5. In vitro tube formation assay 200 ml of Matrigel was added to a 24-well plate and was incubated at 37 C for 30 min to make it coagulate. RF/6 A cells were pre-cultured for 48 h according to each experimental group, and 2 104 cells per well were seeded on 24-well plates with Matrigel and incubated with RPMI 1640 supplemented with 0.5% FBS. Put the transwell chamber with ARPE-19 cells into the 24-well plates which were plated the RF/6 A cells. Then, 24-well plates were placed in a 37 C, 5% CO2 incubator for 24 h. The networks of tubelike structures in each well were observed using an inverted phasecontrast microscope (Olympus, Tokyo, Japan). The experiments were performed for 3 times, and 5 random visual fields from each chamber were counted and averaged. 2.6. Laser-induced mouse CNV model Adult C57BL/6 J (B6) mice were anesthetized by intraperitoneal injection with 0.5% pentobarbital sodium, and the pupils were dilated with tropic amide phenylephrine eye drops (Santen, Osaka, Japan). Laser photocoagulation (647.1 nm; 50 mm spot size; 0.05 s duration; 200 mW) was performed in each mice. Four laser spots were made with a hand-held contact fundus lens (Ocular Instruments, Bellevue, WA) in the 3, 6, 9, and 12 o'clock positions between retinal vessels in the peripapillary area of each eye. If the burns produced a bubble, indicating the broken of Bruch's membrane, and the model is successfully constructed. 2.7. Western blot
Human RPE cell line ARPE-19 was obtained from American Type Culture Collection (ATCC, Manassas, VA). A choroid-retinal endothelial cell line (RF/6 A) from rhesus monkeys was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD) and 1% penicillin/streptomycin (HyClone, UT, USA) at 37 C in 5% CO2. The culture medium was changed every day. The RF/6 A cells were treated with 200 mM cobalt chloride (CoCl2) to the culture medium for 1, 3, 6, 12, and 24 h. Cells without CoCl2 treatment served as the control group.
The proteins and a molecular weight marker were separated on 10% sodium dodecyl sulfateepolyacrylamide gel electrophoresis (SDSePAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. Then, the membranes were incubated with primary antibodies for TBK1 (1:500; Santa Cruz Biotechnology), VEGF (1:500; Santa Cruz Biotechnology) and GAPDH (1:2000; ABclonal) overnight at 4 C. The antibodies were diluted in blocking solution (5% skim milk). Next, the membranes were reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch) at 37 C for 1 h. The blots were then incubated with the chemofluorescent reagent enhanced chemiluminescence (ECL; Thermo Scientific, Rockford, IL) and exposed to X-ray film in the dark.
2.3. TBK1 gene silencing
2.8. Intravitreal injection
TBK1 siRNA (Santa Cruz Biotechnology) was used for transfection according to the manufacturer's instructions. Transfected cells were maintained in RPMI 1640 with 10% FBS for 48 h. Then, the cells were collected to determine the efficiency of TBK1 siRNA by Western blot analysis.
1 ml of anti-TBK1 monoclonal antibody (200 ng/ml) or vehicle (PBS solution) was injected into the vitreous cavity on day 1, and the mice were killed on day 7 after laser photocoagulation. The control mice represented laser-induced CNV without any injection. 2.9. Fundus fluorescein angiography
2.4. In vitro cell proliferation assay 1 105 cells/cm2 RF/6 A cells were seeded in 24-well plates in complete medium and incubated for 24 h. The cells, transfected
The leakage area of CNV was evaluated 7 days after laser photocoagulation by fundus fluorescein angiography (FFA). The development of CNV was examined by using a digital fundus
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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camera connected to a slit-lamp delivery system (Kanghua, Chongqing, China). FFA was performed 4 min after intraperitoneal injection of 0.3 ml of 2% fluorescein sodium (Alcon Laboratories, Irvine, CA). 2.10. Choroidal flat mount The mice were killed after intraperitoneally injection with 0.5% pentobarbital sodium. The entire ocular globes were immediately plated in 4% paraformaldehyde for 1 h. The RPE-choroid-sclera complex eyecups were washed with cold immunocytochemistry buffer (0.5% BSA, 0.2% Tween-20, and 0.1% Triton X-100) in PBS. A 10 mg/ml solution of 40, 60-diamidino-2-phenylindole (DAPI) (1:500), 1 mg/ml solution of Alexa Fluor 568 conjugated isolectinB4 (1:100), and 0.2 U/ml solution of Alexa Fluor 488 conjugated phalloidin (1:100; Invitrogen-Molecular Probes, Eugene, OR) were applied to eyecups overnight at 4 C in blocking solution. The choroidal flat mounts were examined by fluorescence microscopy (LEICA). 2.11. Histopathology For histopathological examination, the mouse eyes were fixed in 4% paraformaldehyde for 3 h. Then, the eyes were enucleated in each group, and 8 mm cryosections were prepared for hematoxylin and eosin (HE) staining on day 7 after laser injury. The sections were cover slipped by mounting medium. Slices with HE staining were observed by using a light microscope (Olympus, Tokyo, Japan).
Fig. 1. Chemical hypoxia increased TBK1 and VEGF expression in RF/6 A cells in vitro. (A) Western blot showd TBK1 and VEGF expression. (B) The histogram showd the densitometric analysis of the average levels of TBK1 and VEGF to GAPDH. *p < 0.05 compared with normal controls. n ¼ 3 in each group.
that tube formation was enhanced. And tube formation was also significantly reduced when TBK1 expression was inhibited (Fig. 2D and E).
2.12. Statistical analysis All values were presented as the mean ± standard deviation (SD). One-way ANOVAs were used for statistical comparisons of multiple groups. Descriptive statistics were performed using Stata statistical software version 11.0 (Stata Corp, College Station, TX). P < 0.05 was considered statistically significant. Each experiment consisted of at least three replicates. 3. Results 3.1. The expression of TBK1 and VEGF was up-regulated in RF/6 A cells under hypoxic condition To explore the function of TBK1 in CNV, we examined the expression of TBK1 and VEGF in RF/6 A cells, which were treated with CoCl2 for different time periods (0, 1, 3, 6, 12 and 24 h). Western blot results showed that the expression of TBK1 and VEGF was up-regulated in a similar time-dependent manner under hypoxic conditions, with a peak effect at 6 h (Fig. 1), suggesting that TBK1 may be associated with VEGF expression. 3.2. Knockdown of TBK1 down-regulated VEGF expression and suppressed the proliferation and tube formation activity of RF/6 A cells TBK1 expression in RF/6 A cells was knocked down by TBK1 siRNA transfection, and the VEGF protein level was evaluated with western blotting. TBK1 siRNA significantly down-regulated the TBK1 protein level in the RF/6 A cells. Accordingly, the VEGF protein level was also down-regulated in the RF/6 A cells in the TBK1 siRNA group compared to the control group (Fig. 2A and B). In the Cell Counting Kit-8 (CCK8) cell proliferation assay, TBK1 siRNA inhibited cell proliferation in the hypoxic RF/6 A cells (Fig. 2C). RF/6 A cells grown in a collagen matrix gel under hypoxic conditions showed
3.3. The expression of TBK1 and VEGF was increased in the mouse CNV model We first determined whether the laser-induced CNV model has been established by fundus fluorescein angiography (FFA) (Fig. 3A). Then we extracted protein of choroid-RPE-retina complex for western blot to examine the expression of TBK1 in CNV. After laser photocoagulation, TBK1 expression was up-regulated and peaked at 7 days after injury, showing a similar time-dependent trend with VEGF (Fig. 3B and C).
3.4. Intravitreal injection of anti-TBK1 monoclonal antibody reduced TBK1 and VEGF expression, and inhibited CNV The TBK1 protein level in the choroid-RPE-retina complex was reduced significantly in the anti-TBK1 injection group compared with the control and PBS injection groups. Furthermore, the expression of VEGF showed a similar trend as TBK1 with anti-TBK1 injection (Fig. 4A and B). The FFA showed that the leakage area of CNV was smaller in the anti-TBK1 injection group than in the PBS injection group on day 7 after laser injury (Fig. 4C). Phalloidin labeling examined actin bundles of RPE cells, forming a tightly packed, uniform hexagonal monolayer. DAPI labeling identified the nuclei. At 7 d, a welldefined radial array of isolectin labeled vessels was visible inside CNV in control and vehicle groups. In the anti-TBK1 injection group, there was reduction of neovascular areas compared to control and vehicle injection groups (Fig. 4D). On day 7 after laser injury, histopathology analysis showed that anti-TBK1 monoclonal antibody treatment remarkly decreased the length and thickness of retinal lesions caused by CNV (Fig. 4E and F).
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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Fig. 2. Knockdown of TBK1 down-regulated VEGF expression and suppressed the proliferation and tube formation activity of RF/6 A cells. (A) TBK1 and VEGF expression was measured by western blot in RF/6 A cells following TBK1 siRNA transfection. (B) The histogram showed the densitometric analysis of the average levels for TBK1 and VEGF to GAPDH. (C) The Line chart showed the proliferation of RF/6 A cells in different groups according to the CCK8 assay. (D) Representative photomicrographs showed tube formation in each group of RF/6 A cells. (E) The average number of closed networks of tubes per field. *p < 0.05, statistically significantly different compared to the respective controls. Values represent means ± SD.
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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Fig. 3. The mouse CNV model was established successfully by laser photocoagulation and the expression of TBK1 and VEGF was up-regulated after CNV formation. (A) Leakage area of CNV was examined by FFA. (B) The protein level of TBK1 and VEGF was detected by western blot. GAPDH was used as the loading control. (C) The histogram showed the densitometric analysis of the average levels of TBK1 and VEGF to GAPDH. *p < 0.05 versus the normal control. Values represent means ± SD.
4. Discussion In this study, we show that TBK1 deficiency inhibits proliferation and tube formation of RF/6 A cells and decreases VEGF expression. Intravitreal injection of anti-TBK1 monoclonal antibody significantly reduces CNV in a murine model of wet AMD. These results indicate that TBK1 might be an important target for AMD treatment. AMD is a multifactorial disease wherein a complex interplay of environmental and genetic factors contributes to its pathogenesis. Although the mechanism of AMD is still largely unclear, inflammation, oxidative stress, and apoptosis have been implicated in this process. Furthermore, hypoxia, abnormal lipid metabolism, immune dysfunction, RPE senescence have also been found to be associated with AMD development [16]. Because of stimulation of various factors, RPE cells undergo metabolic disorders and degeneration, and the function of the phagocytic processing of the extracellular articular membrane of photoreceptors is reduced, resulting in diffuse deposition of abnormal lipid substances on the Bruch's membrane, increasing the thickness of the Bruch's
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membrane and reduced elasticity. It is prone to rupture, so that the RPE cells and the Bruch's membrane are separated. Diffusion thickening of the Bruch's membrane affects the diffusion of oxygen from the choroid to the RPE layer and the retina, resulting in hypoxia. During this process, the inflammatory response also plays a very important role [17e21]. Under ischemic and hypoxic conditions, many cytokines are produced, including HIF-1a and VEGF. VEGF can stimulate endothelial cells to produce NO, induce vasodilation, and increase the permeability of endothelial cells, which facilitates the diffusion of plasma proteins into tissues. Therefore, VEGF plays a very important role in the development of CNV. AntiVEGF therapy has also become the main treatment of AMD [5,22,23]. TBK1, a serine-threonine kinase, belongs to the non-canonical IkB kinases, participates in various cellular pathways related to activation of NF-kB and IRF3. TBK1 acts as a key regulator of angiogenesis, inflammation, oxidative stress, and cell senescence, and may be involved in the pathogenesis of CNV in a large part. Previous studies have found that TBK1 is associated with angiogenesis and can promote HUVEC proliferation [13,14]. Moreover, under hypoxic conditions, TBK1 can regulate the expression of HIF1a via the NF-kB signaling pathway, thereby further regulating the expression of VEGF [24]. Cellular senescence is also an important part of the pathogenesis of AMD, and TBK1 can just induce the cell senescence in ischemic injury [25]. We know that TBK1 has extensive homology (more than 64% sequence identity) and structural similarity with IKK a and IKKb. And previous studies have found that inhibition of IKKb could reduce the formation of neovascularization and the inflammatory reactions, including macrophage infiltration, consequently ameliorated CNV formation [26e28]. These results urge us to think about the relationship between TBK1 and CNV. Inflammation is a complex defense reaction that occurs when a tissue with blood vessels in the body causes damage due to various causes. And CNV is a typical inflammatory disease. TBK1 is a member of the inhibitor kB (IkB) kinase-related kinase family, which is involved in inflammatory pathways related to activation of NF-kB. A lot of reports show that inhibition of NF-kB can reduce angiogenesis, VEGF production and HUVEC tube formation [29e32]. In addition, some studies have also found that oxidative stress could activate NF-kB through reactive oxygen species (ROS), which was to some extent consistent with the pathogenesis of AMD [33,34]. Angiogenin (ANG) is reportedly multifunctional, reducing neovascularization and immune inflammation via inhibition of TBK1 [15,35]. Moreover, TBK1 can also regulate the expression of Akt, which is important in many cellular functions including cell proliferation, migration, metabolism and playing critical and diverse roles in the angiogenesis, vasorelaxation, vascular remodeling and VEGF secretion [36e38]. In our current study, we found that TBK1 could enhance the proliferation and tube formation of RF/6 A cells, and increase VEGF expression. Intravitreal injection of anti-TBK1 monoclonal antibody remarkly attenuated CNV formation. These results are consistent with previous studies. In summary, the present study exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and also verified in both the RF/6 A cells chemical hypoxia model and the laser-induced mouse CNV model. These results may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD.
Conflicts of interest The authors have no conflicts of interest to declare.
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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Fig. 4. Intravitreal injection of anti-TBK1 monoclonal antibody reduced TBK1 and VEGF expression, and inhibited CNV. (A) Western blot showed that TBK1 and VEGF expression after anti-TBK1 monoclonal antibody intravitreal injection. (B) Quantification graphs for TBK1 and VEGF to GAPDH. (C) The FFA analysis of CNV leakage was performed at 7 days after laser photocoagulation. (D) Representative images of choroid flat mounts. (E) HE stains images of CNV. (F) Statistical analysis of data. *p < 0.05. Scale bars ¼ 100 mm.
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo Kaixuan Cui b, 1, Shanshan Zhang c, 1, Xiaojuan Liu d, e, Zhenzhen Yan a, Lili Huang f, Xiaowei Yang a, Rongrong Zhu a, **, Aimin Sang a, * a
Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, 226001, China School of Medicine, Nantong University, Nantong, 226001, Jiangsu, China Nantong University, Nantong, Jiangsu, China d Department of Pathogen Biology, Medical College, Nantong University, Nantong, 226001, Jiangsu, China e Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, 226001, Jiangsu, China f Department of Ophthalmology, The First People's Hospital of Nantong, 226001, Jiangsu Province, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2018 Accepted 1 June 2018 Available online xxx
choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), causes severe vision loss among elderly patients. TANK-binding kinase 1 (TBK1) is a ubiquitously expressed serineethreonine kinase and is found to induce endothelial cells proliferation, represent a novel mediator of tumor angiogenesis and exert pro-inflammatory effect. However, the role of TBK1 in choroidal neovascularization has not been investigated so far. In this study, we found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model. Silencing of TBK1 suppressed the proliferation and tube formation activity of RF/6 A cells. Intravitreal injection of anti-TBK1 monoclonal antibody ameliorates CNV formation. Taken together, these findings exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD. © 2018 Elsevier Inc. All rights reserved.
Keywords: Choroidal neovascularization (CNV) Age-related macular degeneration (AMD) TANK-Binding kinase 1 (TBK1) Vascular endothelial growth factor (VEGF)
1. Introduction Visual impairment is a public health problem that affects patients' quality of life. Among all causes of visual impairment, agerelated macular degeneration (AMD) is a leading cause of adult blindness in industrialized countries [1]. A study forecasted that in 2020, global projected cases of any AMD would be 196 million, rising to 288 million in 2040, with the largest number of cases in Asia [2]. Early AMD is characterized by the presence of drusen, pigmentary changes at the macula, without vision loss. There are two forms of advanced AMD, which is associated with vision loss. One is atrophic form (dry AMD), which is characterized by regional retinal pigment epithelium (RPE) loss and eventual degeneration of
* Corresponding author. Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, 226001, China ** Corresponding author. Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, 226001, China E-mail addresses: [email protected] (R. Zhu), [email protected] (A. Sang). 1 These authors contributed equally to this study.
overlying photoreceptors. The other is neovascular form (wet AMD), which is characterized by choroidal neovascularization (CNV). CNV is the growth of blood vessels from the choroid through Bruch's membrane toward the retina, and it is responsible for approximately 80% of cases of severe vision loss due to AMD [3e5]. So far, the treatment of CNV remains one of the hot spots and difficulties in the field of ophthalmology. Vascular endothelial growth factor (VEGF) plays an important role in the pathophysiological process of neovascular AMD (nAMD). Anti-VEGF therapy is currently the standard of care for nAMD [6]. However, this treatment is still controversial and some patients are non-responsive or refractory to anti-VEGF therapy. Moreover, long-term anti-VEGF therapies may not prevent disease progression even in those patients who showed an initial beneficial therapeutic response, and may lead to detrimental effects on retinal function [7,8]. Therefore, we urgently need to find some molecules to regulate the expression and function of VEGF in order to treat AMD. TANK-binding kinase 1 (TBK1), also known as NF-kB-activating kinase (NAK) or T2K, is a ubiquitously expressed serineethreonine
https://doi.org/10.1016/j.bbrc.2018.06.003 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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kinase and it belongs to the non-canonical IkB kinases (IKKs) recognized for its critical role in regulating type I interferon (IFN) production, IFN regulatory factor 3 (IRF3) and NF-kB signaling pathways. TBK1 is an 84 kDa, 729-amino acid protein containing an N-terminal kinase domain, an ubiquitin like domain and two Cterminal coiled coil domains. As a non-canonical IKK, TBK1 is structurally similar to IKKε (another IKK-related kinase) and the canonical IKKs, IKKa and IKKb. It can be activated by viral infections, inflammatory mediators and so on. Previous research found that TBK1 is involved in the activation of various cellular pathways and pro-inflammatory cytokine production, and homeostatic cellular functions such as cell growth and proliferation [9e12]. Moreover, TBK1 induces human umbilical vein endothelial cells (HUVECs) proliferation, represents a novel mediator of tumor angiogenesis and exerts pro-inflammatory effects via upregulation of inflammatory cytokines. However, the role of TBK1 in CNV has not been investigated so far although several data hint at important TBK1 functions, particularly in angiogenesis and inflammation [13e15]. In the present study, we investigated the effects of TBK1 on the development of CNV, and explored the molecular mechanism. We found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model, and knockdown of TBK1 alleviates CNV formation via inhibiting VEGF expression. Our results indicate that TBK1 plays a critical role in the development of CNV and suggest TBK1 may be a potential therapeutic target for human AMD treatment. 2. Materials and methods 2.1. Animals All experimental steps were strictly performed in accordance with the requirements of the Animal Welfare Committee of Nantong University. This study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures for the use of animals were approved by the Center for Laboratory Animals of Nantong University (Nantong, Jiangsu, China). 2.2. Cell culture
with TBK1 siRNA and scramble siRNA, were placed in the wells. Medium with 200 mM CoCl2 was added and changed every 2 days. At the end of each time point, 20 ml of MTT (Sigma, St. Louis, MO) was added into each well for 4 h, then the supernatant was discarded, and 150 ml dimethyl sulfoxide (DMSO) was added for 15 min. The absorbance at 570 nm was recorded using a Microplate Reader (Model 680, Bio-Rad, Hercules, CA). The experiments were repeated at least in triplicate. 2.5. In vitro tube formation assay 200 ml of Matrigel was added to a 24-well plate and was incubated at 37 C for 30 min to make it coagulate. RF/6 A cells were pre-cultured for 48 h according to each experimental group, and 2 104 cells per well were seeded on 24-well plates with Matrigel and incubated with RPMI 1640 supplemented with 0.5% FBS. Put the transwell chamber with ARPE-19 cells into the 24-well plates which were plated the RF/6 A cells. Then, 24-well plates were placed in a 37 C, 5% CO2 incubator for 24 h. The networks of tubelike structures in each well were observed using an inverted phasecontrast microscope (Olympus, Tokyo, Japan). The experiments were performed for 3 times, and 5 random visual fields from each chamber were counted and averaged. 2.6. Laser-induced mouse CNV model Adult C57BL/6 J (B6) mice were anesthetized by intraperitoneal injection with 0.5% pentobarbital sodium, and the pupils were dilated with tropic amide phenylephrine eye drops (Santen, Osaka, Japan). Laser photocoagulation (647.1 nm; 50 mm spot size; 0.05 s duration; 200 mW) was performed in each mice. Four laser spots were made with a hand-held contact fundus lens (Ocular Instruments, Bellevue, WA) in the 3, 6, 9, and 12 o'clock positions between retinal vessels in the peripapillary area of each eye. If the burns produced a bubble, indicating the broken of Bruch's membrane, and the model is successfully constructed. 2.7. Western blot
Human RPE cell line ARPE-19 was obtained from American Type Culture Collection (ATCC, Manassas, VA). A choroid-retinal endothelial cell line (RF/6 A) from rhesus monkeys was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD) and 1% penicillin/streptomycin (HyClone, UT, USA) at 37 C in 5% CO2. The culture medium was changed every day. The RF/6 A cells were treated with 200 mM cobalt chloride (CoCl2) to the culture medium for 1, 3, 6, 12, and 24 h. Cells without CoCl2 treatment served as the control group.
The proteins and a molecular weight marker were separated on 10% sodium dodecyl sulfateepolyacrylamide gel electrophoresis (SDSePAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. Then, the membranes were incubated with primary antibodies for TBK1 (1:500; Santa Cruz Biotechnology), VEGF (1:500; Santa Cruz Biotechnology) and GAPDH (1:2000; ABclonal) overnight at 4 C. The antibodies were diluted in blocking solution (5% skim milk). Next, the membranes were reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch) at 37 C for 1 h. The blots were then incubated with the chemofluorescent reagent enhanced chemiluminescence (ECL; Thermo Scientific, Rockford, IL) and exposed to X-ray film in the dark.
2.3. TBK1 gene silencing
2.8. Intravitreal injection
TBK1 siRNA (Santa Cruz Biotechnology) was used for transfection according to the manufacturer's instructions. Transfected cells were maintained in RPMI 1640 with 10% FBS for 48 h. Then, the cells were collected to determine the efficiency of TBK1 siRNA by Western blot analysis.
1 ml of anti-TBK1 monoclonal antibody (200 ng/ml) or vehicle (PBS solution) was injected into the vitreous cavity on day 1, and the mice were killed on day 7 after laser photocoagulation. The control mice represented laser-induced CNV without any injection. 2.9. Fundus fluorescein angiography
2.4. In vitro cell proliferation assay 1 105 cells/cm2 RF/6 A cells were seeded in 24-well plates in complete medium and incubated for 24 h. The cells, transfected
The leakage area of CNV was evaluated 7 days after laser photocoagulation by fundus fluorescein angiography (FFA). The development of CNV was examined by using a digital fundus
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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camera connected to a slit-lamp delivery system (Kanghua, Chongqing, China). FFA was performed 4 min after intraperitoneal injection of 0.3 ml of 2% fluorescein sodium (Alcon Laboratories, Irvine, CA). 2.10. Choroidal flat mount The mice were killed after intraperitoneally injection with 0.5% pentobarbital sodium. The entire ocular globes were immediately plated in 4% paraformaldehyde for 1 h. The RPE-choroid-sclera complex eyecups were washed with cold immunocytochemistry buffer (0.5% BSA, 0.2% Tween-20, and 0.1% Triton X-100) in PBS. A 10 mg/ml solution of 40, 60-diamidino-2-phenylindole (DAPI) (1:500), 1 mg/ml solution of Alexa Fluor 568 conjugated isolectinB4 (1:100), and 0.2 U/ml solution of Alexa Fluor 488 conjugated phalloidin (1:100; Invitrogen-Molecular Probes, Eugene, OR) were applied to eyecups overnight at 4 C in blocking solution. The choroidal flat mounts were examined by fluorescence microscopy (LEICA). 2.11. Histopathology For histopathological examination, the mouse eyes were fixed in 4% paraformaldehyde for 3 h. Then, the eyes were enucleated in each group, and 8 mm cryosections were prepared for hematoxylin and eosin (HE) staining on day 7 after laser injury. The sections were cover slipped by mounting medium. Slices with HE staining were observed by using a light microscope (Olympus, Tokyo, Japan).
Fig. 1. Chemical hypoxia increased TBK1 and VEGF expression in RF/6 A cells in vitro. (A) Western blot showd TBK1 and VEGF expression. (B) The histogram showd the densitometric analysis of the average levels of TBK1 and VEGF to GAPDH. *p < 0.05 compared with normal controls. n ¼ 3 in each group.
that tube formation was enhanced. And tube formation was also significantly reduced when TBK1 expression was inhibited (Fig. 2D and E).
2.12. Statistical analysis All values were presented as the mean ± standard deviation (SD). One-way ANOVAs were used for statistical comparisons of multiple groups. Descriptive statistics were performed using Stata statistical software version 11.0 (Stata Corp, College Station, TX). P < 0.05 was considered statistically significant. Each experiment consisted of at least three replicates. 3. Results 3.1. The expression of TBK1 and VEGF was up-regulated in RF/6 A cells under hypoxic condition To explore the function of TBK1 in CNV, we examined the expression of TBK1 and VEGF in RF/6 A cells, which were treated with CoCl2 for different time periods (0, 1, 3, 6, 12 and 24 h). Western blot results showed that the expression of TBK1 and VEGF was up-regulated in a similar time-dependent manner under hypoxic conditions, with a peak effect at 6 h (Fig. 1), suggesting that TBK1 may be associated with VEGF expression. 3.2. Knockdown of TBK1 down-regulated VEGF expression and suppressed the proliferation and tube formation activity of RF/6 A cells TBK1 expression in RF/6 A cells was knocked down by TBK1 siRNA transfection, and the VEGF protein level was evaluated with western blotting. TBK1 siRNA significantly down-regulated the TBK1 protein level in the RF/6 A cells. Accordingly, the VEGF protein level was also down-regulated in the RF/6 A cells in the TBK1 siRNA group compared to the control group (Fig. 2A and B). In the Cell Counting Kit-8 (CCK8) cell proliferation assay, TBK1 siRNA inhibited cell proliferation in the hypoxic RF/6 A cells (Fig. 2C). RF/6 A cells grown in a collagen matrix gel under hypoxic conditions showed
3.3. The expression of TBK1 and VEGF was increased in the mouse CNV model We first determined whether the laser-induced CNV model has been established by fundus fluorescein angiography (FFA) (Fig. 3A). Then we extracted protein of choroid-RPE-retina complex for western blot to examine the expression of TBK1 in CNV. After laser photocoagulation, TBK1 expression was up-regulated and peaked at 7 days after injury, showing a similar time-dependent trend with VEGF (Fig. 3B and C).
3.4. Intravitreal injection of anti-TBK1 monoclonal antibody reduced TBK1 and VEGF expression, and inhibited CNV The TBK1 protein level in the choroid-RPE-retina complex was reduced significantly in the anti-TBK1 injection group compared with the control and PBS injection groups. Furthermore, the expression of VEGF showed a similar trend as TBK1 with anti-TBK1 injection (Fig. 4A and B). The FFA showed that the leakage area of CNV was smaller in the anti-TBK1 injection group than in the PBS injection group on day 7 after laser injury (Fig. 4C). Phalloidin labeling examined actin bundles of RPE cells, forming a tightly packed, uniform hexagonal monolayer. DAPI labeling identified the nuclei. At 7 d, a welldefined radial array of isolectin labeled vessels was visible inside CNV in control and vehicle groups. In the anti-TBK1 injection group, there was reduction of neovascular areas compared to control and vehicle injection groups (Fig. 4D). On day 7 after laser injury, histopathology analysis showed that anti-TBK1 monoclonal antibody treatment remarkly decreased the length and thickness of retinal lesions caused by CNV (Fig. 4E and F).
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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Fig. 2. Knockdown of TBK1 down-regulated VEGF expression and suppressed the proliferation and tube formation activity of RF/6 A cells. (A) TBK1 and VEGF expression was measured by western blot in RF/6 A cells following TBK1 siRNA transfection. (B) The histogram showed the densitometric analysis of the average levels for TBK1 and VEGF to GAPDH. (C) The Line chart showed the proliferation of RF/6 A cells in different groups according to the CCK8 assay. (D) Representative photomicrographs showed tube formation in each group of RF/6 A cells. (E) The average number of closed networks of tubes per field. *p < 0.05, statistically significantly different compared to the respective controls. Values represent means ± SD.
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
K. Cui et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
Fig. 3. The mouse CNV model was established successfully by laser photocoagulation and the expression of TBK1 and VEGF was up-regulated after CNV formation. (A) Leakage area of CNV was examined by FFA. (B) The protein level of TBK1 and VEGF was detected by western blot. GAPDH was used as the loading control. (C) The histogram showed the densitometric analysis of the average levels of TBK1 and VEGF to GAPDH. *p < 0.05 versus the normal control. Values represent means ± SD.
4. Discussion In this study, we show that TBK1 deficiency inhibits proliferation and tube formation of RF/6 A cells and decreases VEGF expression. Intravitreal injection of anti-TBK1 monoclonal antibody significantly reduces CNV in a murine model of wet AMD. These results indicate that TBK1 might be an important target for AMD treatment. AMD is a multifactorial disease wherein a complex interplay of environmental and genetic factors contributes to its pathogenesis. Although the mechanism of AMD is still largely unclear, inflammation, oxidative stress, and apoptosis have been implicated in this process. Furthermore, hypoxia, abnormal lipid metabolism, immune dysfunction, RPE senescence have also been found to be associated with AMD development [16]. Because of stimulation of various factors, RPE cells undergo metabolic disorders and degeneration, and the function of the phagocytic processing of the extracellular articular membrane of photoreceptors is reduced, resulting in diffuse deposition of abnormal lipid substances on the Bruch's membrane, increasing the thickness of the Bruch's
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membrane and reduced elasticity. It is prone to rupture, so that the RPE cells and the Bruch's membrane are separated. Diffusion thickening of the Bruch's membrane affects the diffusion of oxygen from the choroid to the RPE layer and the retina, resulting in hypoxia. During this process, the inflammatory response also plays a very important role [17e21]. Under ischemic and hypoxic conditions, many cytokines are produced, including HIF-1a and VEGF. VEGF can stimulate endothelial cells to produce NO, induce vasodilation, and increase the permeability of endothelial cells, which facilitates the diffusion of plasma proteins into tissues. Therefore, VEGF plays a very important role in the development of CNV. AntiVEGF therapy has also become the main treatment of AMD [5,22,23]. TBK1, a serine-threonine kinase, belongs to the non-canonical IkB kinases, participates in various cellular pathways related to activation of NF-kB and IRF3. TBK1 acts as a key regulator of angiogenesis, inflammation, oxidative stress, and cell senescence, and may be involved in the pathogenesis of CNV in a large part. Previous studies have found that TBK1 is associated with angiogenesis and can promote HUVEC proliferation [13,14]. Moreover, under hypoxic conditions, TBK1 can regulate the expression of HIF1a via the NF-kB signaling pathway, thereby further regulating the expression of VEGF [24]. Cellular senescence is also an important part of the pathogenesis of AMD, and TBK1 can just induce the cell senescence in ischemic injury [25]. We know that TBK1 has extensive homology (more than 64% sequence identity) and structural similarity with IKK a and IKKb. And previous studies have found that inhibition of IKKb could reduce the formation of neovascularization and the inflammatory reactions, including macrophage infiltration, consequently ameliorated CNV formation [26e28]. These results urge us to think about the relationship between TBK1 and CNV. Inflammation is a complex defense reaction that occurs when a tissue with blood vessels in the body causes damage due to various causes. And CNV is a typical inflammatory disease. TBK1 is a member of the inhibitor kB (IkB) kinase-related kinase family, which is involved in inflammatory pathways related to activation of NF-kB. A lot of reports show that inhibition of NF-kB can reduce angiogenesis, VEGF production and HUVEC tube formation [29e32]. In addition, some studies have also found that oxidative stress could activate NF-kB through reactive oxygen species (ROS), which was to some extent consistent with the pathogenesis of AMD [33,34]. Angiogenin (ANG) is reportedly multifunctional, reducing neovascularization and immune inflammation via inhibition of TBK1 [15,35]. Moreover, TBK1 can also regulate the expression of Akt, which is important in many cellular functions including cell proliferation, migration, metabolism and playing critical and diverse roles in the angiogenesis, vasorelaxation, vascular remodeling and VEGF secretion [36e38]. In our current study, we found that TBK1 could enhance the proliferation and tube formation of RF/6 A cells, and increase VEGF expression. Intravitreal injection of anti-TBK1 monoclonal antibody remarkly attenuated CNV formation. These results are consistent with previous studies. In summary, the present study exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and also verified in both the RF/6 A cells chemical hypoxia model and the laser-induced mouse CNV model. These results may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD.
Conflicts of interest The authors have no conflicts of interest to declare.
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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Fig. 4. Intravitreal injection of anti-TBK1 monoclonal antibody reduced TBK1 and VEGF expression, and inhibited CNV. (A) Western blot showed that TBK1 and VEGF expression after anti-TBK1 monoclonal antibody intravitreal injection. (B) Quantification graphs for TBK1 and VEGF to GAPDH. (C) The FFA analysis of CNV leakage was performed at 7 days after laser photocoagulation. (D) Representative images of choroid flat mounts. (E) HE stains images of CNV. (F) Statistical analysis of data. *p < 0.05. Scale bars ¼ 100 mm.
Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003
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Please cite this article in press as: K. Cui, et al., Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.003