Mol Cell Biochem (2015) 398:123–134 DOI 10.1007/s11010-014-2212-2

Effects of p75 neurotrophin receptor on regulating hypoxiainduced angiogenic factors in retinal pigment epithelial cells Jingjing Zhang • Jun Zhao • Yujing Bai • Lvzhen Huang • Wenzhen Yu • Xiaoxin Li

Received: 22 June 2014 / Accepted: 31 August 2014 / Published online: 9 September 2014 Ó Springer Science+Business Media New York 2014

Abstract Retinal pigment epithelium (RPE) exerts critical roles in the maintenance of the normal functions of the retina, whereas RPE dysfunction can induce retina neovascularization. p75 neurotrophin receptor (p75NTR) has been shown to play essential roles in angiogenesis. However, the function of p75NTR in the RPE remains unclear. In the present study, we demonstrated that p75NTR was highly expressed in the human choroidal neovascularization membranes. For in vitro study, RPE was exposed to hypoxia, and a knockdown of p75NTR was achieved via lentivirus-mediated RNA interference. The results showed that hypoxia induced the expression of p75NTR in the RPE, and the knockdown of p75NTR rescued RPE proliferation activity and inhibited apoptosis which induced by hypoxia. After the deletion of p75NTR, RPE-secreted pro-angiogenic factors (vascular endothelial growth factor and plateletderived growth factor), inflammatory factors [interleukin 1 beta (IL1b), IL18, and stromal cell-derived factor 1], and matrix metalloproteinases (MMPs) (MMP3 and MMP9) were down-regulated under hypoxic conditions. While the RPE secreted anti-angiogenic factors (pigment epitheliumderived factor) and angiostatin, the tissue inhibitors of Jingjing Zhang and Jun Zhao contributed equally to this paper and are co-first authors. J. Zhang  Y. Bai (&)  L. Huang  W. Yu  X. Li Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People’s Hospital, Xizhimen South Street 11, Xi Cheng District, Beijing 100044, China e-mail: [email protected] J. Zhao Department of Ophthalmology, Linyi People’s Hospital, Linyi, Shandong, China

metalloproteinases (TIMPs) (TIMP-1 and TIMP-3) were up-regulated after the knockdown of p75NTR. The human umbilical vein endothelial tube formation ability can be inhibited when it is co-cultured with the supernatant extract from p75NTR-knockdown RPE under hypoxic induction. These results suggest that the knockdown of p75NTR suppressed pro-angiogenic factors which induced by hypoxia while promoting the anti-angiogenesis-related factors in the RPE. It is indicated that p75NTR could be a potential therapeutic target for RPE hypoxia or oxidative stress diseases. Keywords p75 neurotrophin receptor (p75NTR)  Retinal pigment epithelial (RPE)  Angiogenesis  Hypoxia

Introduction The retina is a light-sensitive layer that lines the back of the eye [1]. The retinal pigment epithelium (RPE), which is interposed between the neural retina and the choroid, plays critical roles in the maintenance of the normal functions of the retina [2]. Among the multiple functions of the RPE layer are the absorption of light, maintenance of the visual cycle, phagocytosis of photoreceptor outer membranes, secretion, and immune privileges [2]. RPE dysfunction has been implicated as the cause of many retinal diseases, including retinitis pigmentosa and age-related macular degeneration (AMD) [3, 4]. Choroidal neovascularization (CNV) is a serious complication of exudative AMD (or wet-AMD), which can lead to significant loss of central vision [5]. Although several pathological mechanisms underlying the formation and progression of CNV remain to be fully elucidated, recent studies have implicated an unbalanced secretion of pro-

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and anti-angiogenesis regulators of RPE as key steps [5]. Pro-angiogenesis regulators include vascular endothelial growth factor (VEGF), angiopoietins, basic-fibroblast growth factor, and other inflammation factors, such as interleukins and chemokines [6]. The anti-angiogenesis regulators include pigment epithelium-derived factor (PEDF), tissue inhibitors of matrix metalloproteinases (TIMPs), and endostatins [6]. Until recently, VEGF antagonists such as Ranibizumab and Aflibercept were used as the standard treatment for inhibiting the progression of CNV [7]. However, these treatment strategies are palliative, and patients must subject to these repeated treatments throughout their lives. Due to the importance of the RPE’s role in the progression of CNV, restoring the normal function of the RPE could represent a new treatment target. p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor superfamily, has been shown to play essential roles in regulating a variety of functions in the developing and adult nervous system [8]. p75NTR can mediate cell survival or death and can facilitate or attenuate proliferation, depending on the cellular context [8]. The function of p75NTR has mainly been studied in neurons [9]. However, recent studies have indicated that p75NTR contributes to angiogenesis [10] and exerts critical roles in retinal inflammation in the rodent diabetic retina [11]. Other studies have reported that the p75NTR levels increased dramatically in dystrophic retinas, particularly in the photoreceptor layer and RPE layer [12]. Nevertheless, the actual role of p75NTR in RPE is not known. In the present study, we aimed to examine the effects of p75NTR in hypoxia-induced, angiogenesis-related factors in RPE. Our results suggested that the knockdown of p75NTR suppressed the expression of the hypoxia-induced, proangiogenic factors while up-regulating the anti-angiogenic factors. The deletion of p75NTR in the RPE can also inhibit HUVEC tube formation when it is co-cultured in hypoxic conditions. These promising results indicated that p75NTR could be a potential therapeutic target for RPE hypoxia or oxidative stress-related diseases, such as CNV.

Materials and methods Immunohistochemistry assays in CNV patients membranes Three human CNV sub-retinal membranes were surgically excised from patients after proper consent and approval. Immunohistochemistry (IHC) was performed with 6 lm frozen serially sections across the whole specimen. Briefly,

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tissue sections were fixed in 4 % paraformaldehyde and blocked with 10 % normal goat serum. Anti-p75NTR antibody (Abcam, ab8874, 1:200 dilution, Abcam Inc., Cambridge, MA, USA) was applied to the tissue sections at 4 °C overnight. Sections were incubated with the primary antibody for 18 h at 4 °C and sections incubated with PBS served as controls. Antibody was visualized by the avidin– biotin-immunoperoxidase complex (ABC) method (ABC Vectastain reagent) and followed by horseradish peroxidase complex with 3,30 -diaminobenzidine tetrahydrochloride (Dako, Glostrp, Denmark) staining. For each case, normal rabbit IgG was used as a negative control. Images of slides were viewed and captured on a Nikon microscope (Eclipse E800). Cell cultures Human RPE cells [ARPE19 cell line, American Type Culture Collection (ATCC), CRL-2302, Manassas, VA, USA] and human umbilical vein endothelial cells (HUVECs, ATCC, CRL-1730, Manassas, VA, USA) were used in this study, as previously described [13, 14]. Cells were cultured in DMEM/F12 medium (HyClone, Hyclone, Grand Island, NY, USA) containing 10 % fetal bovine serum (FBS, HyClone, Hyclone, Grand Island, NY, USA). All of the cells used were harvested between passage 3 and passage 7. RPE cells were exposed to hypoxia (1 % oxygen) for 24 or 48 h. Lentivirus-mediated shRNA knockdown of p75NTR expression The knockdown of p75NTR was induced by a lentivirusmediated RNA interference vector (TL311178, OriGene Technologies, Rockville, MD, USA). The following small interfering RNA (siRNA) target sequences in the human p75NTR gene were selected: #1, 50 -CCTACATAGCC TTCAAGAGGTGGAACAGC-30 ; #2, 50 -CAGAACAAGA CCTCATAGCCAGCACGGTG-30 ; #3, 50 -CTCCTGCCAG GACAAGCAGAACACCGTGT-30 ; and #4, 50 -ACCGA GCCGTGCAAGCCGTGCACCGAGTG-30 . A scrambled sequence (50 -GCACTACCAGAGCTAACTCAGATAGTA CT-30 ) was used as negative control. For lentivirus transduction, RPE cells were sub-cultured at 5 9 104 cells/well into 6-well culture plates. After grown to 40 % confluence, cells were transducted with shRNA lentivirus at a multiplicity of infection (MOI) of 50. Cells were harvested at 24 and 48 h after infection, and the knockdown efficiency of p75NTR was evaluated by real-time RT-PCR and Western blot analysis. All transfections were carried out using the Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, USA).

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RNA extraction and real-time PCR of p75NTR RPE cells were exposed to normoxia and hypoxia for 24 h. The total RNA of the RPE cells treated with the lentivirusmediated RNA interference vector were lysed in Trizol, according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Total RNA was used for reverse transcription and amplified by quantitative real-time PCR, as described previously [13]. The primers used for the real-time PCR were p75NTR: forward 50 -TGCACAGCGACAGTGGCATC-30 ; reverse 50 -TCCTCACGCTTGGTCAGGGG-30 ; and GAPDH: forward 50 -GAGTCCACTGGCGTCTTCAC30 ; reverse 50 -GTTCACACCCATGACGAACA-30 . Each experiment was repeated five times. RPE cell proliferation assays A BrdU assay (#6813, Cell Signaling Technology, Danvers, MA, USA) was used for evaluation RPE cell proliferation as previously described [15] and read by an ELISA microplate reader (Finstruments Multiskan Models 347; MTX Lab Systems, Inc., Vienna, VA, USA). RPE cells were seeded in 96-well plates and incubated for 24 and 48 h under hypoxia. Each experiment was performed in five wells and repeated at least three times. Flow cytometric analysis of RPE apoptosis The apoptosis study was performed according to the manufacturer’s instructions (FITC Annexin V Apoptosis Detection Kit; BD Biosciences, San Diego, CA, USA), as previously reported [13]. Briefly, 1 9 106 RPE cells were seeded in 6-well plates and incubated for 48 h under hypoxic culture conditions. Then, the cells were detached with EDTA, and stained with Annexin-V-FITC and propidium iodide (PI), according to the manufacturers’ instructions. Flow cytometry analysis was immediately performed (ex/em = 488/530 nm). Then, 104 cells were collected and divided into four groups: dead cells (Annexin V-/PI?, UL), late apoptotic cells (Annexin V?/PI?, UR), viable cells (Annexin V-/PI-, LL), and early apoptotic cells (Annexin V?/PI-, LR). The samples were analyzed by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) with Cell Quest software (BD, Biosciences). The apoptotic rate was calculated as the percentage of early apoptotic cells (LR) and late apoptotic cells (UR) compared with the total cells. Western blot analysis RPE cells were treated by a lentivirus-mediated RNA interference vector and cultured under hypoxic conditions for 48 h. Total cell lysates were prepared using protein

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extraction and protease inhibitor kits (Pierce, Rockford, IL, USA). After centrifugation, the supernatant was collected and analyzed by immunoblots. Equal amounts of protein (30 lg) were separated by 10 % SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Amersham, Little Chalfont, UK). Immunoblots were probed with primary and secondary antibodies, and immunoreactive bands were visualized with enhanced chemiluminescence Western blot detection reagents (Pierce). For digital quantification, the membranes were analyzed using the ImageJ software. The loading was controlled using antibodies against b-actin. The Western blot analyses were repeated three times, and qualitatively similar results were obtained. The primary antibodies used included the following: antip75 NGF receptor antibody (Abcam, ab8874, 1:1,000), antiVEGF 165A [6B7] antibody (Abcam, ab69479, 1:1,000), anti-PDGF BB antibody (Abcam, ab23914, 1:1,000), antiPEDF antibody (Abcam, ab180711, 1:1,000), anti-Angiostatin antibody (Abcam, ab93060, 1:1,000), anti-SDF1 (D32F9) antibody (Cell Signaling Technology, #3530, 1:1,000), anti-IL18 antibody (Abcam, ab137664, 1:500), antiIL1 beta antibody (Abcam, ab2105, 1:500), anti-MMP3 antibody [SPM293] (Abcam, ab17790, 1:1,000), anti-MMP9 antibody [EP1254] (Abcam, ab76003, 1:1,000), anti-TIMP1 antibody (Abcam, ab38978, 1:1,000), and anti-TIMP3 antibody (Abcam, ab85926, 1:1,000). For sequential blotting with additional antibodies, the membranes were stripped with stripping buffer and re-probed with the indicated antibodies. HUVECs tube formation study HUVECs tube formation study was used for in vitro evaluation of angiogenesis, which is a convenient and quantifiable assay to test the vascular formation properties [13]. This assay measures the ability of endothelial cells forming capillary-like structures. In this study, RPE cells were cultured under hypoxia, with or without p75NTR interference, for 48 h. Then, the supernatants were collected and used as HUVECs culture medium immediately. For tube formation assay, 150 ll of Matrigel (BD Biosciences, San Diego, CA, USA) was poured into 48-well plates and then incubated at 37 °C for 30 min. The HUVECs (5 9 104 per well) incubated in the collected supernatants were seeded on the Matrigel and cultured for 8 h. Upon plating with the extracellular matrix (ECM) support, HUVECs attach and generate mechanical forces on the surrounding extracellular support matrix to create tracks that facilitate forming hollow lumens. The length of the capillary-like structures in two-dimensional networks in Matrigel was measured from five randomly chosen fields using the Image J software. The experiments were repeated three times.

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Fig. 1 p75NTR expression in CNV membranes by immunohistochemistry. a Represents the negative control of CNV membrane staining for p75NTR which showed no obvious expression

(2009 magnification); b represents p75NTR is highly expressed in CNV membrane and cytoplasm (2009 magnification); c and d represent the enlarged images from a and b (4009 magnification)

Statistical analysis Data analysis was performed using the statistical software Prism 5 (GraphPad Software Inc., San Diego, CA, USA). All data are presented as the mean ± SEM. Differences were evaluated using Student’s t test for pair-wise comparisons. p \ 0.05 was considered a statistically significant difference.

membrane we excised was a fibrotic membrane tissue, whereas the membrane was not a vascular membrane. As shown in Fig. 1, p75NTR is highly expressed and localized throughout the sub-retinal membranes from patients with CNV. No visible staining of p75NTR was observed in the negative control membranes. These results indicated that p75NTR is a component of CNV sub-retinal membranes.

Results

Expression of p75NTR after RNA interference

p75NTR was highly expressed in the CNV membranes as detected by immunohistochemistry

The knockdown of p75NTR was induced by a lentivirus-mediated RNA interference vector (shRNAs #3) in RPE cells, and the deletion efficiency was evaluated. As shown in Fig. 1, hypoxia can induce the expression of p75NTR significantly compared with normoxic conditions. After RNA interference, the expression of p75NTR was inhibited at both the RNA (Fig. 2a) and protein (Fig. 2b, c) levels under normoxic and hypoxic conditions.

To evaluate the expression of p75NTR in the in vivo tissue, the human CNV sub-retinal membranes were used. Because only in the condition of vascular tissue rupture, which leads to retinal hemorrhage and fibrous membrane proliferation, the patients will undergo the surgical treatment. Thus, the

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Fig. 2 Expression of p75NTR in RPE cells after knockdown by lentivirus-mediated RNA interference. a and b show the results and statistical analysis of real-time PCR and Western blots for p75NTR expression in the RPE after the knockdown of p75NTR. c Shows the immunoblot image of p75NTR expression after the knockdown of p75NTR. The lentivirus-mediated RNA interference vector can

efficiently inhibit the expression of p75NTR. All of the results were compared with a normoxic control group, and the results are stated as the ratio fold. Real-time PCR analyses were repeated five times, and Western blot analyses were repeated three times; qualitatively similar results were obtained for both. Data are presented as the mean ± SEM **p \ 0.01; ***p \ 0.0001

Fig. 3 RPE cell proliferation assay in hypoxic conditions after the knockdown of p75NTR. a and b Show the results and statistical analysis of RPE cell proliferation activity at different time points (24 h (a), 48 h (b)). The knockdown of p75NTR rescued cell

proliferation activity under the hypoxic culture condition. The experiment was performed in six wells and repeated at least three times. The data are presented as the mean ± SEM. *p \ 0.05; **p \ 0.01

p75NTR deletion rescued RPE proliferation activity under hypoxia

proliferation activity when cultured in hypoxia condition compared to the hypoxia control groups (p \ 0.05).

The BrdU Assay was used to evaluate the RPE cell proliferation in vitro. The results showed that hypoxia inhibited RPE cell proliferation time-dependently from 24 to 48 h and had a significant difference compared with the control groups (Fig. 3). p75NTR deletion rescued RPE

p75NTR deletion inhibited hypoxia-induced RPE apoptosis A flow cytometry test was used to evaluate the effects of p75NTR deletion on RPE cell apoptosis (early apoptosis and late

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Fig. 4 Apoptosis study of RPE cells in hypoxic conditions after the knockdown of p75NTR. Apoptosis was quantified by flow cytometry measured by Annexin V and PI staining. a (normoxia condition), b (hypoxia condition) and c (hypoxia culture condition with p75NTR deletion) show representative images of the apoptosis examination. d Shows the results and statistical analysis of RPE apoptosis under

hypoxic conditions after the knockdown of p75NTR. UR: late apoptotic cells; LR: early apoptotic cells; UR ? LR: total apoptotic cells. The experiment was repeated at least 3 independent times. DMEM ? 10 %FBS control was set to 100 %. The data are presented as the mean ± SEM. *p \ 0.05; **p \ 0.01

apoptosis). In the present study, hypoxia induced RPE apoptosis significantly after incubation for 48 h compared with the normal control group (p \ 0.01). p75NTR RNA interference inhibited RPE apoptosis under hypoxia-induced conditions at both time points (Fig. 4).

is an endogenous anti-angiogenic protein. Immunoblot analysis showed that hypoxia induced the up-regulation of pro-angiogenic factors (VEGF and PDGF) while down-regulating anti-angiogenic factors (PEDF and angiostatin) expression as expected. However, p75 NTR RNA interference restored the imbalance of pro- and anti-angiogenesis factors, to a certain extent (Fig. 5).

Pro- and anti-angiogenic factors in p75NTR-knockdown RPE cells under hypoxia Angiogenic factors were key regulators for vascular formation and progression. In the present study, we measured the levels of pro-angiogenic factors (VEGF and PDGF) and anti-angiogenic factors (PEDF and angiostatin) in p75NTR-deleted RPE cells under hypoxic conditions. VEGF is believed to be the most potent factor in the induction of angiogenesis, whereas PEDF

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Inflammatory cytokines in p75NTR-knockdown RPE cells under hypoxia Inflammatory cytokines such as SDF1, IL1b, and IL18 were reported to be related to angiogenesis [6], and the relation between p75NTR and these factors is studied in the present study. Immunoblot analysis revealed that hypoxia induced the

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Fig. 5 Expression of pro- and anti-angiogenic factors in RPE cells under hypoxic conditions after the knockdown of p75NTR. Immunoblot images (a) and statistical analyses (b and c) for the expression of pro- and antiangiogenic factors are shown. The knockdown of p75NTR restored the imbalance of proand anti-angiogenesis factors in RPE under the hypoxic culture conditions. The Western blot analyses were repeated three times, and qualitatively similar results were obtained. The data are presented as the mean ± SEM. *p \ 0.05

expression of the above factors significantly comparing to normal control, whereas p75NTR RNA interference inhibited the expression of SDF1, IL1 b, and IL18 alike under hypoxia condition comparing to control group (Fig. 6a, b). Matrix metalloproteinases and their inhibitors in p75NTR-knockdown RPE cells under hypoxia Matrix metalloproteinases and their inhibitors are important for Bruch’s membrane degeneration, which facilitates the formation of CNV. Figure 7 shows that under hypoxic culture conditions, MMP3 and MMP9 were up-regulated

(Fig. 7a, b), whereas their inhibitors, TIMP1 and TIMP3, were down-regulated (Fig. 7a, c). Similar to the patterns of pro- and anti-angiogenic factors, after the deletion of p75NTR, the expression patterns of MMPs and TIMPs were reversed compared with those of the hypoxic control group. p75NTR deletion inhibited co-cultured HUVECs tube formation The Matrigel tube formation assay is a quantifiable method of testing the angiogenic/anti-angiogenic properties of compounds on vascular endothelial cells in vitro by measuring the ability of

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Fig. 6 Inflammatory cytokine expression of the RPE in hypoxic condition after the knockdown of p75NTR. Immunoblot images (a) and statistical analyses (b) for SDF1, IL1 b, and IL18 expressions are shown. The knockdown of p75NTR inhibited the inflammatory cytokine expression in RPE under the hypoxic culture conditions. The Western blot analyses were repeated three times, and qualitatively similar results were obtained. The data are presented as the mean ± SEM. *p \ 0.05

endothelial cells form capillary-like structures. In the present study, HUVECs had an impaired capacity to form a regular network in the p75NTR RNA interference cell supernatant coculture group after the RPE were exposed to hypoxic conditions (Fig. 8c). The length of the angiogenic network showed a significant difference compared with the hypoxic controls (Fig. 8).

Discussion The p75 neurotrophin receptor (p75NTR) was the first identified receptor for nerve growth factor (NGF) [16]. p75NTR is expressed on a variety of cell types and can influence many cellular functions [17]. Previously, the effects of p75NTR were primarily studied on neural cells, and p75NTR was reported to play an ambiguous role, either killing or stimulating neural cell survival and differentiation [8, 18]. However, the function of this receptor in other cell types has remained enigmatic [3, 11, 19]. Recently, the function of p75NTR in angiogenesis has been reported. Caporali A and colleagues reported that p75NTR expression impairs the cell activity of cultured ECs and endothelial progenitor cells (EPCs), inhibiting angiogenesis in vitro and in vivo through the VEGF-A/Akt/eNOS/NO pathway [10]. After that study, Tahiri et al. reported that the down-

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regulation of p75NTR expression significantly blocked the antiangiogenic effects of T-lymphocyte-derived microparticles in a choroidal angiogenesis model [19]. In the present study, we focused on the function of p75NTR in the RPE cell, which plays critical roles in the maintenance of retinal function. The results showed that hypoxia induced the expression of p75NTR in RPE cells and that the knockdown of p75NTR rescued RPE cell proliferation activity and inhibited RPE apoptosis which induced by hypoxia. After the deletion of p75NTR, the RPE secreted fewer pro-angiogenic factors (VEGF and PDGF) and inflammatory factors (IL1b, IL18, and SDF-1) and downregulated the secretion of matrix metalloproteinases (MMP3 and MMP9) in hypoxic conditions. Conversely, the secretions of anti-angiogenesis factors (PEDF and angiostatin) and metalloproteinases inhibitor tissue inhibitors (TIMP-1 and TIMP-3) were up-regulated. These changes in the expression of the aforementioned factors impaired the ability of HUVECs to form tubes. Moreover, p75NTR was highly expressed in the human CNV membranes as detected by immunohistochemistry, which further verified that p75NTR involved in the formation and progression of retina diseases. These results indicated that p75NTR could act as a potential therapeutic target for RPE hypoxia or oxidative damage diseases, including AMD. To

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Fig. 7 Matrix metalloproteinases and inhibitory factor expression of RPE in hypoxic conditions after the knockdown of p75NTR. Immunoblot images (a) and statistical analyses (b and c) for the matrix metalloproteinase and inhibitor expressions are shown. The knockdown of p75NTR down-regulated MMP3 and MMP9 while up-regulating TIMP1 and TIMP3 in the RPE under the hypoxic culture conditions. The Western blot analyses were repeated three times, and qualitatively similar results were obtained. The data are presented as the mean ± SEM. *p \ 0.05

our knowledge, this is the first study to address the function of p75NTR in the RPE. The RPE is a differentiated, cuboidal monolayer of cells with a complex supportive role in visual function [5, 20]. The failure of any one of these functions can lead to the development of retinal degenerative diseases, including AMD [2]. ‘Wet’ or neovascular AMD (nAMD) is a severe type of AMD, with CNV serving as its defining characteristic [5]. CNV is a pathological condition in which abnormal choroidal blood vessels grow through Bruch’s membrane, penetrate the RPE

layer and extend into the subretinal space [6]. Consequently, the blood vessels leak fluid, leading to serous retinal detachment and ultimately inducing retinal hemorrhage, which causes vision loss [21]. Although the exact pathophysiology of CNV is not known, several molecules (including VEGF, MMP, and PEDF), the breakdown of the blood-retina barrier comprising RPE and Bruch’s membrane, and several inflammatory processes precede the development of CNV [22]. During the formation and progression of CNV, abnormal RPE plays a central role [22, 23].

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Fig. 8 Tube formation study of HUVECs in the supernatant of p75NTR knockdown RPE. Representative images of the tube formation assay of different groups: a normoxic culture condition supernatant group; b hypoxic culture condition supernatant group; c hypoxic culture condition with p75NTR deletion supernatant group;

d statistical analysis of HUVECs’ tube formation. All of the pictures (a–c) were taken using a 1009 magnification under a Zeiss light microscope. All of the data were measured at least three times and are presented as the mean ± SEM. *p \ 0.05, **p \ 0.01

In the normal retina, the pro- and anti-angiogenic factors are in balance with one another. When the correct balance is disturbed by pathological events, an angiogenic switch occurs [24]. The aging process and chronic oxidative stress lead to an imbalance in the secretion of protective and toxic factors in the RPE [2]. As we have shown in the present study, hypoxia-induced RPE dysfunction facilities CNV formation at least in three aspects: (1) up-regulated angiogenic factors; (2) induction of inflammatory expression, and (3) promotion of ECM degradation. Increased levels of VEGF and PDGF could act on vascular endothelial cells with various effects, including increasing vascular permeability, inducing angiogenesis, and promoting endothelial cell proliferation [25, 26]. Conversely, PEDF and angiostatin are endogenous angiogenesis inhibitors that can counter these pro-angiogenesis effects. The increased expression of interleukins and chemokines can exacerbate RPE injury on the one hand and prompt macrophage accumulation on the other, further stimulating CNV development [27]. Recently, several articles reported

that IL1b and IL18 are related to AMD, IL1b inhibition prevents CNV, while IL18 attenuates experimental CNV [28–30]. Here we found that p75NTR inhibition reduced the expression of IL1b and IL18 in RPE under hypoxia culture condition, while the exact mechanisms need to be explored in further studies. Additionally, the introduction of an imbalance in the matrix proteolysis system (MMPs and TIMPs) is also involved in rebuilding the ECM in angiogenesis [5]. The deletion of p75NTR not only restores such imbalances in angiogenesis-related factors but also inhibits HUVECs’ tube formation when they are co-cultured with the p75NTR interference RPE supernatant. In the present study, we also found that p75NTR down-regulation is related to rescue cell proliferation ability and attenuate apoptosis procedure. And the mechanisms could be: (1) up-regulation of PEDF, which is a neurotrophic factors; (2) inhibition of inflammatory factors, such as IL1b, IL18; (3) down-regulation of chemokines, such as SDF-1, which will induce aggregation of macrophage and other lymphocytes.

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Previously, Caporali A concluded that p75NTR inhibits endothelia cell (EC) cell cycle progression and impairs EC ability to form capillary-like tubular structures in vitro [10]. Conversely, they also demonstrated that p75NTR expression inhibits reparative neovascularization in ischemic limb muscles in vivo [10]. The findings of p75NTR exert the function on EC seem to contradict with our study, which we found that p75NTR deletion could be beneficial for angiogenesis inhibition. However, the differences between the two studies probably because of the treatment targets are completely different, i.e. the previous paper performed by Caporali A used EC, whereas we used RPE cells in the present study. The second reason may also because the complex interactions between the in vivo tissues, and the different response of p75NTR neurotrophin receptor. As known, the development of CNV is a complex process that first comprises RPE dysfunction and moves to the induction of choroidal neovascular ingrowth. Therefore, in the present study our treatment target was primarily the restoration of RPE function. Having thus normalized the RPE, we hoped that the abnormal angiogenesis would degenerate. In addition, the two articles have a number of common findings, including that p75NTR overexpression is deleterious to the cell expressing its receptor. For example, in Caporali A’s paper, the overexpression of p75NTR was found to induce EC apoptosis, allowing them to establish angiogenic degeneration after p75NTR overexpression. However, we found that when p75NTR is overexpressed in the RPE, the ultimate result is cell death. Our primary object was to rescue the potential function of such cells, restoring them to a normal state. Currently, the standard of care for treating nAMD and other vascular retinal diseases is anti-VEGF treatment with VEGF antagonists, such as Ranibizumab and Aflibercept. In our study, we attempted to restore the function of RPE by inhibiting p75NTR, which can inhibit angiogenesisrelated factors and induce the expression of anti-angiogenesis factors, returning the RPE to its normal physiological conditions. By restoring the expression patterns of pro- and anti-angiogenesis, angiogenesis itself can be halted. Our findings probably have important implications and may help in the design of innovative therapeutic approaches targeting CNV. During our study, we recognized as a possible limitation that we have not investigated whether RPE-specific p75NTR deletion is beneficial for CNV in an in vivo model. Thus, further studies with laser-induced CNV models in P75NTR knockout mice may constitute a useful animal model for evaluating potential therapeutic methods for the treatment of nAMD. Acknowledgments We thank Bin Wang for her help with the FACS detection. This work was supported by the National Natural Science

133 Foundation of China Grant (81200690), the National Basic Research Program of China (973 Program, 2011CB510200), and the Peking University People’s Hospital Research and Development Fund (RDB2012-24). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Conflict of interest interests exist.

The authors have declared that no conflict of

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Effects of p75 neurotrophin receptor on regulating hypoxia-induced angiogenic factors in retinal pigment epithelial cells.

Retinal pigment epithelium (RPE) exerts critical roles in the maintenance of the normal functions of the retina, whereas RPE dysfunction can induce re...
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