March 2012
Volume 53, Issue 3
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Retinal Cell Biology  |   March 2012
Complement and UV-Irradiated Photoreceptor Outer Segments Increase the Cytokine Secretion by Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • Katharina Lueck
    From the Ophtha-Lab,
  • Maren Hennig
    From the Ophtha-Lab,
  • Albrecht Lommatzsch
    Department of Ophthalmology, St. Franziskus Hospital, Muenster, Germany.
  • Daniel Pauleikhoff
    From the Ophtha-Lab,
    Department of Ophthalmology, St. Franziskus Hospital, Muenster, Germany.
  • Susanne Wasmuth
    From the Ophtha-Lab,
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1406-1413. doi:10.1167/iovs.11-8889
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      Katharina Lueck, Maren Hennig, Albrecht Lommatzsch, Daniel Pauleikhoff, Susanne Wasmuth; Complement and UV-Irradiated Photoreceptor Outer Segments Increase the Cytokine Secretion by Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(3):1406-1413. doi: 10.1167/iovs.11-8889.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Age-related macular degeneration (AMD) is accompanied by increased complement activation, and by lipofuscin accumulation in retinal pigment epithelial (RPE) cells due to incomplete degradation of photoreceptor outer segments (POS). The influence of POS, ultraviolet (UV)-irradiated POS and human complement sera (HCS) on cytokine secretion from RPE cells was therefore examined.

Methods.: RPE cells were incubated with POS or UV-POS every other day for 1 week. The autofluorescence (AF) was measured photometrically and by flow cytometry. Senescence-associated genes were analyzed by RT-PCR. Internalization and degradation of POS were determined using phagocytosis and degradation assays, and lysosomal function by neutral red uptake. RPE cells in polycarbonate cell culture inserts were incubated apically with POS or UV-POS and afterward basally with HCS. C7-deficient HCS was used as control. The integrity of the cell monolayer was assessed by measuring the transepithelial electrical resistance (TER) and the permeability. Interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and vascular endothelial growth factor were quantified by ELISA.

Results.: POS treatment led to an increased AF and senescence marker expression, which were further elevated in response to UV-POS. UV-POS were preferentially accumulated over POS and the lysosomal function was impaired due to UV-POS. HCS intensified the cytokine production compared with controls. POS had no effect, though UV-POS combined with HCS induced a significant increase in all cytokines.

Conclusions.: RPE cultivation with UV-POS might serve as a model to investigate the accumulation of lipofuscin-like structures. The enhanced cytokine secretion due to UV-POS with HCS may account for an increased susceptibility for lipofuscin-loaded cells to complement, inducing a proinflammatory environment as observed in AMD.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells, exhibiting a polarized morphology. 1 To the basolateral side, the Bruch's membrane (BrM) separates the RPE from the choroidea, whereas apically, the photoreceptor tips are embedded in the RPE membrane. The RPE maintains several functions such as the directed transport of nutrients, disposal of deposited material and secretion of immune-mediating cytokines. 2 Moreover, RPE cells serve a protective function through the production of melanin, which captures free radicals generated by light exposure. 3 Another essential function comprises the phagocytosis of shed photoreceptor outer segments (POS) and their degradation through the lysosomal system. 4  
RPE cells are at particular risk for oxidative damage due to their lifelong exposure to light and their oxygen-rich environment. 5 POS are rich in polyunsaturated fatty acids, explaining their high susceptibility to oxidation processes. 6 Lipid peroxidation of POS induces conformational changes which prevent their interaction with lysosomal enzymes, leading to incomplete degradation and accumulation of POS remnants in RPE cells. 7 The exposure of POS to ultraviolet (UV) light was previously described to transform POS into lipofuscin-like structures, which accumulate when fed to RPE cell cultures. 8  
Lipofuscin accumulation is associated with RPE aging processes. 9 It is composed of proteins, lipids, and autofluorescent material with the major component bis-retinoid A2E mainly derived from POS membranes. 10 Lipofuscin accumulation is accompanied by a diminished capacity to respond to stress, altered photoreceptor function, and enhanced phagocytosis. 11 This altered cell metabolism may play a role in dysfunction and loss of RPE and photoreceptor cells, contributing to degenerative diseases including age-related macular degeneration (AMD). 12,13  
AMD characterizes the progressive degeneration of RPE and photoreceptor cells with advancing age. 14 The dry course of AMD, hallmarked by drusen, can lead to geographic atrophy of RPE cells. 15 Exudative AMD is designated by the generation of choroidal neovascularization (CNV), eliciting visual loss in a large proportion of patients. 16,17  
Drusen are extracellular deposits located between the basal lamina of the RPE and the innermost layer of BrM. 18 The presence of drusen is a significant indicator for early AMD 19,20 and for the development of neovascular membranes. 21 The characterization of drusen revealed proteins involved in inflammatory processes including several complement components. 15 The role of complement in AMD was further elucidated when a single nucleotide polymorphism (SNP) in the complement factor H (CFH) gene was found in approximately half of AMD patients. 22 24 This SNP is associated with an increased activation of complement. The complement system as an important part of innate immunity constitutes a first, nonspecific defense against potential pathogens. It provides a cascade of proteins, whose activation leads to the assembly of C5b-9 on surfaces of target cells to induce cell lysis. However, sublytic amounts of C5b-9 are capable of activating several cell populations and exerting chemotactic activity. 25,26  
AMD is regarded as a chronic subclinical inflammatory disease. 27,28 Local inflammatory responses in the RPE may be conducted through the secretion of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1). The expression of IL-6 was observed to be elevated in laser-induced murine CNV, 29 and IL-8 concentration in aqueous humor has been associated with the CNV severity. 30 The aqueous MCP-1 concentration in patients with exudative AMD has been correlated with the occurrence of macula edema. 31 Vascular endothelial growth factor (VEGF) participates in angiogenic processes and intensifies vascular permeability. 32 VEGF is produced by the RPE and contributes essentially to the development and maintenance of the choriocapillaris. 33 Increasing amounts, however, were associated with abnormal vessel growth within exudative AMD. 34  
In this study, RPE cells were incubated with UV-irradiated (UV)-POS to induce the accumulation of structurally altered POS, similar to lipofuscin-like structures in RPE cells in vivo. We analyzed autofluorescence (AF) and expression of senescence-associated genes apolipoprotein J (Apo J), fibronectin, osteonectin, and transgelin (SM22), which are considered markers for cellular age. 35,36 We also investigated the effect of human complement sera (HCS) on the polarized secretion of IL-6, IL-8, MCP-1, and VEGF by untreated, POS-, or UV-POS-treated RPE cells. 
Methods
Isolation of POS
POS were isolated from porcine eyes as described previously 37 and stored at −80°C until further use. 
UV-Irradiation of POS
UV-irradiation was performed as previously described, 38 accompanied by a few modifications. POS (1 × 107) were diluted in 1 mL PBS in a 24-well plate and exposed to a UV-light source with a wavelength of 254 nm and 2 × 15 W for 3 hours at a distance of 20 cm. After the irradiation period, POS were thoroughly removed from the well, centrifuged at 6000g for 10 minutes and directly used in experiments. 
Cell Culture and Treatment
Human ARPE-19 cells (ATCC number CRL-2302) 1 were cultivated in Dulbecco's modified Eagle's medium and Ham's F12 1:1 (DMEM/F12; Biochrom, Berlin, Germany) at 37°C with 5% carbon dioxide. The media was supplemented with 100 U/mL streptomycin/penicillin (PAA Laboratories GmbH, Pasching, Austria) and 10% fetal calf serum (FCS). When grown to confluence, cells were incubated with 2 × 107 POS or UV-POS/mL medium and 1% FCS for every other day of a week. The polarized secretion of cytokines was analyzed using RPE cells grown on polycarbonate cell culture inserts (Transwell, #3401, 0.4 μm pore size; Corning, Amsterdam, the Netherlands). 
At 5 weeks postconfluence, when the transepithelial electrical resistance (TER) was stable, a permeability assay was performed to ensure the integrity of the cell monolayer. POS were added to the apical compartment. Subsequent incubation with 2% and 4% HCS was carried out in the basolateral compartment for 24 hours. Medium alone and 4% of the commercially available C7-deficient HCS were used as controls. Unless stated otherwise, all reagents were purchased from Sigma (Steinheim, Germany). 
Reverse Transcriptase (RT)-Polymerase Chain Reaction (PCR)
Total RNA was isolated using a kit (RNeasy Plus Mini Kit; Qiagen, Hildesheim, Germany) following the manufacturer's instructions. Total RNA (0.5 μg) was transcribed into cDNA with a kit (Omniscript RT Kit; Qiagen). The cDNA template was applied to RT-PCR, accomplished with a PCR kit (HotStarTaqPlus Master Mix; Qiagen). The expression of Apo J, SM22, fibronectin, and osteonectin was analyzed with specific primers 39,40 under the following conditions (Table 1) using a PCR thermal cycler (Thermocycler Gradient T; Biometra, Göttingen, Germany). The PCR products were separated by electrophoresis on a 1.5% agarose gel at 100 V. Relative fold change in each cytokine expression was calculated by normalizing values to GAPDH (primer designed by Teresa Hsi, Harvard NeuroDiscovery Center) expression. Expression values of treated samples were compared with respective values of untreated samples. 
Table 1.
 
Primer Sequences and PCR Conditions
Table 1.
 
Primer Sequences and PCR Conditions
Target Sequence from 5′ to 3′ bp Initial Step Denaturation, Annealing, Elongation Number of Cycles Final Elongation
Apo J F gaa atg aag ctg aag gct ttc ccg 286 5′ at 95°C 45 s at 94°C, 45 s at 57°C, 1′ at 72°C 24 10′ at 72°C
B gga act gta aag ctg ggc tat gga
SM22 F tga agg tgc ccg aga acc ca 367 30 s at 94°C, 40 s at 57°C, 30 s at 72°C 35
B atc tgc cga ggt cgt ccg tag c
Fibronectin F tgc caa cct tta cag acc ta 492 30 s at 94°C, 40 s at 57°C, 30 s at 72°C 35
B ctc atc tcc ctc ctc act ca
Osteonectin F gca gag gaa acc gaa gag ga 207 30 s at 94°C, 40 s at 57°C, 30 s at 72°C 35
B ggc aaa gaa gtg gca gga ag
GAPDH F atg aca tca aga agg tgg tg 177 30 s at 94°C, 30 s at 55°C, 1′ at 72°C 25
B cat acc agg aaa tga gct tg
AF Measurement
For photometric measurement of the cellular AF, cells were washed, covered with 100 μL PBS, and analyzed using a plate reader at 490 nm wavelength. For flow cytometry analysis, cells were detached with trypsin-EDTA, resuspended in 100 mM PBS-EDTA, and the AF was analyzed with a flow cytometer (BD FACSCalibur; BD Bioscience, Heidelberg, Germany). The emitted fluorescence of 488 nm for 50,000 events was measured and analyzed with computer software (Win MDI 2.9, freeware created by Joseph Trotter, Scripps Research Institute, La Jolla, CA). 
Phagocytosis Assay
To differentiate between bound and internalized POS, cells were incubated with 0.2% trypan blue for 10 minutes to quench the fluorescence of externally bound POS. 41 PBS control treated cells reflected bound and internalized POS. After excessive washing, the cells were detached and the AF was analyzed via flow cytometry as described above. 
In Situ Distribution of POS
POS were labeled with AlexaFluor 555 (Invitrogen, Darmstadt, Germany) and fed to RPE cells in polycarbonate cell culture inserts (Transwell; Corning). The cells were fixed in 4.5% formaldehyde for 10 minutes, dehydrated in ascending series of alcohol (70%–100%) for 20 minutes each and embedded in paraffin. Paraffin sections of 7 μm were prepared with a microtome (RM 2135; Leica, Wetzlar, Germany), deparaffinized and the nuclei were stained with Hoechst 33342 (10 μg/mL). Cells were covered with 10% glycerol and analyzed with a fluorescent light microscope (BX40; Olympus, Hamburg, Germany). 
POS Degradation Assay
POS and UV-POS treated cells were lysed in 20 mM Tris-HCl (pH 7.5), 140 mM NaCl, 50 mg/mL deoxycholate, 0.1% SDS, 1% Triton X-100, 10% glycerol, 1 mM Na3Vo4, 1 mM DTT, 1 μM pepstatin, 10 μM leupeptin, and 1 mM PMSF. 42 The lysate was incubated on ice for 15 minutes and sonicated for 3 × 20 seconds. The samples were separated on an SDS-PAGE at 120 V and transferred to a nitrocellulose membrane at 350 mA for 50 minutes. The degradation of phagocytosed POS was determined using a mouse monoclonal anti-rhodopsin antibody (clone 1D4; Santa Cruz Biotechnology, Heidelberg, Germany). The detected bands were visualized by chemiluminescent reaction (Western Blotting Luminol Reagent Kit, Santa Cruz Biotechnology) and exposed to x-ray film. Densitometric analysis of obtained bands was performed by software developed by Wayne Rasband (ImageJ; National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html) and the data were normalized to α-tubulin, which was detected with a monoclonal mouse α-tubulin antibody (clone TU-01, 1:5000; Exbio, Vestec, Czech Republic). 
Lysosomal Function
The lysosomal function was analyzed using a neutral red assay. Cells were washed and incubated with 0.005% neutral red for 2 hours until the dye accumulated in the cells. After efficient washing, the internalized dye was released using 50% ethanol and 1% glacial acetic acid. The optical density (OD) was measured at a wavelength of 550 nm. 
TER Measurement
The TER was measured weekly with an epithelial volt-ohm meter (Epithelial Voltohmmeter; World Precision Instruments, Berlin, Germany). Additional measurements were taken before and after the HCS treatment. The TER was calculated by subtracting the value of an insert without cells from the experimentally measured value. This value was multiplied with the surface area of the insert to obtain the resistance of the effective growth area in Ωcm2. 43  
Permeability Assay
The permeability of the RPE cells grown in polycarbonate cell culture inserts (Transwell; Corning) was determined by measuring the movements of sodium fluorescein from the apical to the basolateral compartment. 44 One blank filter without cells was used as a control. The dye (25 μg/mL) was added to the apical side, and 50 μL was collected from the basolateral compartment at each time point (0, 1, 2, 4, and 24 hours). These 50 μL were replaced by equal amounts of medium. The OD of the removed sample was measured with an eight-channel photometer (MRX; Dynatech, Denkendorf, Germany) at a wavelength of 490 nm. 
Enzyme-Linked Immunosorbent Assay (ELISA)
Cell culture supernatants were harvested after 24 hours, stored at −20°C and analyzed for IL-6, IL-8, MCP-1 (human IL-6/IL-8/MCP-1 OptEIA ELISA Set; BD Bioscience) and VEGF (human VEGF DuoSet; R & D Systems, Wiesbaden, Germany) by sandwich-ELISA according to the manufacturer's protocol. The OD was measured at a wavelength of 450 nm. 
Statistical Analysis
Data were presented as means with standard deviations. One-way ANOVA and Tukey post hoc were performed for normally distributed data to analyze the differences between more than two groups. P < 0.05 was considered statistically significant, marked with an asterisk (*). 
Results
POS and UV-POS Increased the Expression of Senescence-Associated Genes
Apo J, fibronectin, SM22, and osteonectin were constitutively expressed by RPE cells as detected by RT-PCR. The expression of these senescence-associated genes tended to be elevated in response to POS when compared with untreated cells. The treatment with UV-POS led to a significant rise in the expression of Apo J, fibronectin, osteonectin, and SM22 (Figs. 1A, 1B). 
Figure 1.
 
Increased expression of senescence-associated genes of UV-POS treated RPE cells compared with untreated and POS-treated cells analyzed by RT-PCR (A, B). Data are normalized mean and standard deviations from four independent experiments. * P < 0.05.
Figure 1.
 
Increased expression of senescence-associated genes of UV-POS treated RPE cells compared with untreated and POS-treated cells analyzed by RT-PCR (A, B). Data are normalized mean and standard deviations from four independent experiments. * P < 0.05.
Elevated Intracellular AF after UV-POS Treatment
POS-treated RPE cells revealed an increased AF measured photometrically at a wavelength of 490 nm compared with untreated cells. The incubation with UV-POS led to an additional significant increase in AF (Fig. 2A). Flow cytometry analysis demonstrated a constitutive fluorescence at a wavelength of 488 nm for untreated RPE cells and a geo mean at 4.1 ± 2.3. The POS treatment revealed a shift toward an increased AF compared with untreated cells and an increased geo mean (9.6 ± 1.3). The incubation with UV-POS represented an additional rise in the geo mean (16.8 ± 1.7), accompanied by an additional RPE cell population when analyzed by flow cytometry (Fig. 2B). To distinguish whether this enhanced AF is based on extracellular binding or internalization of POS or UV-POS, the intracellular AF was measured. We recorded a shift toward an elevated intracellular AF due to POS, verified by a rise in the geo mean from 5.2 ± 1.1 to 9.7 ± 1.1 (P < 0.001). A further increase in the intracellular AF was observed using UV-POS treated RPE cells (geo mean: 12.7 ± 1.5; P < 0.001) (Fig. 2C). Moreover, the geo mean of the total AF of UV-POS treated cells was higher than the intracellular AF (Figs. 2B, 2C). 
Figure 2.
 
Elevated total and intracellular AF of UV-POS treated RPE cells compared with untreated and POS treated cells. (A) The total AF was determined photometrically at a wavelength of 490 nm. Presented is the mean out of 16 separate measurements. (B) Representative histogram of the total AF from four different experiments measured by flow cytometry at a wavelength of 488 nm. (C) Histogram of the intracellular AF, representing internalized POS and UV-POS, analyzed by flow cytometry. Untreated, gray histogram; POS, open gray histogram; UV-POS, open black histogram.
Figure 2.
 
Elevated total and intracellular AF of UV-POS treated RPE cells compared with untreated and POS treated cells. (A) The total AF was determined photometrically at a wavelength of 490 nm. Presented is the mean out of 16 separate measurements. (B) Representative histogram of the total AF from four different experiments measured by flow cytometry at a wavelength of 488 nm. (C) Histogram of the intracellular AF, representing internalized POS and UV-POS, analyzed by flow cytometry. Untreated, gray histogram; POS, open gray histogram; UV-POS, open black histogram.
Increased Accumulation of UV-POS in RPE Cells
Paraffin embedded ARPE-19 cells, incubated apically with AlexaFluor 555–labeled POS, revealed sporadic accumulation of POS in the cells (Fig. 3A). In comparison, incubation with UV-POS revealed an increased accumulation within cells and an increased binding to the cell surface (Fig. 3B). Proteins samples analyzed by Western blot revealed distinct bands at 40 kDa corresponding to the POS constituent rhodopsin. POS treated ARPE-19 cells showed a slightly thinner band and a decrease in rhodopsin approximately 44% in comparison with UV-POS treated cells (Figs. 3C, 3D). 
Figure 3.
 
Increased accumulation of UV-POS in RPE cells. Representative Hoechst staining (blue) of paraffin sections prepared of RPE cells grown on polycarbonate cell-culture inserts (Transwell; Corning), incubated apically with labeled (red) (A) POS and (B) UV-POS. (C) Representative Western blot analysis results for rhodopsin using cell lysates of POS and UV-POS treated cells demonstrating a decreased degradation of UV-POS. (D) Densitometric analysis of rhodopsin Western blot bands (n = 5) normalized to α-tubulin. *P < 0.05.
Figure 3.
 
Increased accumulation of UV-POS in RPE cells. Representative Hoechst staining (blue) of paraffin sections prepared of RPE cells grown on polycarbonate cell-culture inserts (Transwell; Corning), incubated apically with labeled (red) (A) POS and (B) UV-POS. (C) Representative Western blot analysis results for rhodopsin using cell lysates of POS and UV-POS treated cells demonstrating a decreased degradation of UV-POS. (D) Densitometric analysis of rhodopsin Western blot bands (n = 5) normalized to α-tubulin. *P < 0.05.
Decreased Neutral Red Uptake in Response to UV-POS
The RPE cell viability, measured by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, was not altered by POS, UV-POS, or HCS treatment (data not shown). A neutral red assay was performed to determine the lysosomal function. Incubation of RPE cells with POS had no significant impact on the neutral red uptake. Treatment with UV-POS, however, revealed a significant decline in the lysosomal incorporation of the dye (Fig. 4). 
Figure 4.
 
Decreased neutral red uptake through UV-POS treated RPE cells compared with untreated or POS-treated cells. Illustrated are mean and standard deviations from eight independent experiments. *P < 0.05.
Figure 4.
 
Decreased neutral red uptake through UV-POS treated RPE cells compared with untreated or POS-treated cells. Illustrated are mean and standard deviations from eight independent experiments. *P < 0.05.
Integrity of a Dense Cell Monolayer
The time-dependent increase of the TER reflected a saturation curve. After 5 weeks the TER was stable at 65 ± 8 Ωcm2 (Fig. 5A). Permeability assays revealed intact RPE cell monolayer. Increasing amounts of the apically added sodium fluorescein were detected basally from blank polycarbonate cell culture inserts (Transwell; Corning) without cells. Approximately 6 μg/mL of the dye passed the permeable insert quickly during the first 4 hours. The cell monolayer however retained the fluorescent protein for at least 24 hours, the time period of the HCS treatment (Fig. 5B). 
Figure 5.
 
Verification of a dense cell monolayer. (A) TER measurements and (B) permeability assay on RPE cells grown on polycarbonate cell culture inserts (Transwell; Corning) or inserts without cells as a control. (A) The TER was measured once a week. Mean and standard deviations were evaluated from triplicate measurements from 10 inserts. (B) Apical to basal movement of sodium fluorescein was determined at different time points. Presented are mean and SD of representative duplicate tests.
Figure 5.
 
Verification of a dense cell monolayer. (A) TER measurements and (B) permeability assay on RPE cells grown on polycarbonate cell culture inserts (Transwell; Corning) or inserts without cells as a control. (A) The TER was measured once a week. Mean and standard deviations were evaluated from triplicate measurements from 10 inserts. (B) Apical to basal movement of sodium fluorescein was determined at different time points. Presented are mean and SD of representative duplicate tests.
Decreased TER in Response to UV-POS Combined with HCS
The TER was not impaired in response to C7-deficient HCS, HCS, or POS incubation. The TER we observed due to UV-POS and combined treatment with UV-POS and C7-deficient HCS tended to be lower compared with that of untreated or POS treated cells. However, a significant reduction in the TER was detected as a result of UV-POS and HCS in combination (Fig. 6). 
Figure 6.
 
Decreased TER of ARPE-19 cells grown on polycarbonate cell culture inserts (Transwell; Corning) after incubation with UV-POS and HCS compared with untreated, POS, or C7-deficient HCS treated cells. Presented are mean and SD from four independent experiments. *P < 0.05.
Figure 6.
 
Decreased TER of ARPE-19 cells grown on polycarbonate cell culture inserts (Transwell; Corning) after incubation with UV-POS and HCS compared with untreated, POS, or C7-deficient HCS treated cells. Presented are mean and SD from four independent experiments. *P < 0.05.
Enhanced Basal Secretion of Cytokines due to UV-POS with HCS
Untreated ARPE-19 cells cultured in polycarbonate cell culture inserts (Transwell; Corning) produced IL-6, IL-8, MCP-1, and VEGF constitutively. The addition of HCS led to a significant increase in the cytokine secretion, independent of any pretreatment with POS or UV-POS. C7-deficient HCS revealed only minor effects in this respect. POS and UV-POS incubation did not alter the cytokine production compared with untreated cells. However, secretion of IL-6 (P < 0.001), IL-8 (P = 0.015), MCP-1 (P = 0.001), and VEGF (P < 0.001) was strongly elevated into the basal compartment due to UV-POS/4% HCS compared with POS/4% HCS (Figs. 7A–D). The apical secretion of these cytokines was also increased due to HCS. However, we observed no significant elevation in the secretion in response to UV-POS with HCS (data not shown). 
Figure 7.
 
IL-6 (A), IL-8 (B), MCP-1 (C), and VEGF (D) secretion toward the basal side by untreated, POS, or UV-POS treated RPE cells, as well as in addition with C7-deficient HCS and HCS. Shown are mean and SD from four separate experiments collected by ELISA. *P < 0.05.
Figure 7.
 
IL-6 (A), IL-8 (B), MCP-1 (C), and VEGF (D) secretion toward the basal side by untreated, POS, or UV-POS treated RPE cells, as well as in addition with C7-deficient HCS and HCS. Shown are mean and SD from four separate experiments collected by ELISA. *P < 0.05.
Discussion
Cellular aging is a complex and protracted process associated with the progressive accumulation of detrimental changes. 11 This process is characterized by the aggregation of debris, waste products, and lipofuscin. Underlying mechanisms such as light damage, oxidative stress, lipid peroxidation, and modifications in life-supporting organelles like mitochondria and lysosomes are also seen during AMD pathogenesis. Build-up of lipofuscin within RPE cells can cause degeneration leading to photoreceptor degeneration and the development of AMD. 7,45  
We observed a significant increase in the expression of Apo J, fibronectin, osteonectin, and SM22 after incubation with UV-POS. Consistent with this, the expression of these senescence-associated genes has been found upregulated by RPE cells or human diploid fibroblasts by hydrogen peroxide or transforming growth factor-ß1. 46 The extracellular matrix (ECM) component fibronectin and Apo J have been determined constituents of drusen in AMD patients, 19,47 and raised amounts of Apo J were observed in a couple of apoptosis models. 48 Osteonectin and SM22 participate in the ECM turnover, cell proliferation, and in senescence-associated changes. 49 Given that overexpression of senescence-associated genes indicates a criterion for stress-induced premature senescence, the increase in Apo J, fibronectin, osteonectin, and SM22 may imply that treatment with UV-irradiated POS induce RPE cell aging. 
Our results showed an increase in the total AF in response to POS treatment and a further significant elevation after treatment with UV-POS. The accumulation of autofluorescent material has previously been found in the cytoplasm of postconfluent RPE cell cultures. 50 The bis-retinoid fluorophore A2E, the main component of lipofuscin, 51 was visualized by spectrophotometric fundus AF imaging, 52 and increased lipofuscin fluorescence was observed at the edges of areas with geographic atrophy in AMD eyes. 53 We further detected an increase in the intracellular AF, indicating a preferred accumulation of UV-POS over POS. Moreover, the difference in the geo mean between total and intracellular AF of UV-POS treated cells may give evidence for increased external binding of UV-POS. The elevated AF of RPE cells fed with UV-POS as well as the increased intracellular deposition of UV-POS may reflect the elevated accumulation of structurally altered POS. Up to 33% of the RPE cytoplasmic space was shown to be occupied by lipofuscin from the age of 70 onward. 54 The higher amount of rhodopsin in UV-POS treated cells suggested a decrease in UV-POS degradation, which may support the hypothesis of the accumulation of altered POS as lipofuscin-like structures. 
We further found a significant decrease in the neutral red uptake by UV-POS-treated RPE cells, possibly reflecting an impaired lysosomal function. The RPE lysosome is responsible for the terminal degradation of overaged proteins and accumulating waste products. Lysosomes of aged postmitotic cells become enlarged and more lipofuscin-loaded with advancing age, 55 and are predominantly affected by senescence processes. 56,57 The inability of the lysosomal compartment to completely degrade proteins contributes toward lipofuscin accumulation. 12 Accumulation of the lipofuscin component A2E was investigated to decrease the effectiveness of lysosomal enzymes via increase in the lysosomal pH. A2E also inhibits the degradation of POS and contributes toward blue light-mediated tearing of the lysosomal membranes. 58 60 The impaired lysosomal function due to UV-POS implicates the accumulation of structurally altered POS in RPE cells as a result of incomplete degradation. Therefore, the ARPE-19 cell culture treatment with UV-POS might represent a model to investigate senescent and lipofuscin-loaded RPE cells. 
To mimic the morphologic polarization of RPE cells in the human eye in vivo and to evaluate the directed secretion of mediators, we cultured ARPE-19 cells in polycarbonate cell culture inserts (Transwell; Corning) as previously described. 1 We found a stable barrier function of RPE cells as indicated by sodium fluorescein retention and a constant TER, before experiments. 
We measured a significant decline in the TER due to UV-POS combined with HCS, suggesting an impaired integrity of the cell monolayer. Recent findings demonstrated a decreased TER in RPE cells treated with hydrogen peroxide and complement-sufficient serum. 61 HCS only had an impact on the barrier function, when a second influencing parameter affected the cells. This indicated UV-POS damaged cells to be more susceptible to HCS, arguing for complement-mediated attack exerting a significantly stronger influence on dysfunctional RPE cells. 
Our experiments revealed an increase in the IL-6, IL-8, MCP-1, and VEGF secretion toward the basal side after HCS treatment, independent of the pretreatment with POS or UV-POS. In previous studies we demonstrated an elevated production of the aforementioned cytokines by RPE cells in response to sublytic C5b-9 formation. 62 A directed secretion toward the basal side concerning these mediators has also been described. 63 The basolateral increase in IL-6, IL-8, and MCP-1 by RPE cells may generate a proinflammatory environment and attract immune cells. Elevated amounts of VEGF potentially favor new vessel growth from the choroidea into the neuroretina. Therefore, HCS may contribute to unspecific inflammatory and angiogenic processes as seen in AMD. In contrast, C7-deficient HCS administrated only minor effects, which could be attributed to unspecifically activated RPE cells by various proteins in the serum. In this regard, thrombin stimulated the IL-6 and MCP-1 release by human adipocytes, 64 and C5a, generated during complement activation, increased IL-6, IL-8, and MCP-1 in primary human RPE cells. 65  
We found no impact of POS or UV-POS treatment on the cytokine release. Contrary to that, other investigators revealed elevated amounts of IL-8 and MCP-1 in response to UV-POS, though the UV-irradiation was performed under a longer time period, a lower radiation force, and the POS concentration fed to the cell culture was half compared with our experiments. 38 However, our study revealed a significant increase in IL-6, IL-8, MCP-1, and VEGF by RPE cells treated with UV-POS combined with HCS. 
Regarding this outcome, RPE cells appear to be affected by complement activation, though deleterious effects mediated through complement might only be achieved in addition of a secondary stimulus. These data provide evidence for an enhanced susceptibility for senescent and lipofuscin-loaded RPE cells to complement, which could be relevant during AMD development. 
Footnotes
 Supported by Akademie des Sehens and Voltmann Foundation.
Footnotes
 Disclosure: K. Lueck, None; M. Hennig, None; A. Lommatzsch, None; D. Pauleikhoff, None; S. Wasmuth, None
The authors thank Martin Busch at Ophtha-Lab, Department of Ophthalmology, Muenster, for his help in processing the statistical data and Jennifer Williams for proofreading and helpful comments. 
References
Dunn KC Aotaki-Keen AE Putkey FR Hjelmeland LM . ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res. 1996;62:155–169. [CrossRef] [PubMed]
Hollborn M Kohen L Wiedemann P Enzmann V . The influence of pro-inflammatory cytokines on human retinal pigment epithelium cell receptors. Graefes Arch Clin Exp Ophthalmol. 2001;239:294–301. [CrossRef] [PubMed]
Sarna T Burke JM Korytowski W . Loss of melanin from human RPE with aging: possible of melanin photooxidation. Exp Eye Res. 2003;76:89–98. [CrossRef] [PubMed]
Bok D . The retinal pigment epithelium: a versatile partner in vision. J Cell Sci. 1993;17:189–195. [CrossRef]
Zarbin MA . Current concepts in the pathogenesis of age-related macular degeneration. Arch Ophthalmol. 2004;122:598–614. [CrossRef] [PubMed]
Katz ML Gao CL Rice LM . Formation of lipofuscin-like fluorophores by reaction of retinal with photoreceptor outer segments and liposomes. Mech Ageing Dev. 1996;92:159–174. [CrossRef] [PubMed]
Weiter JJ Delori FC Wing GL Fitch KA . Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes. Invest Ophthalmol Vis Sci. 1986;27:145–152. [PubMed]
Whilmark U Wrigstad A Roberg K Brunk UT Nilsson SE . Formation of lipofuscin in cultured retinal pigment epithelial cells exposed to pre-oxidized photoreceptor outer segments. APMIS. 1996;104:272–279. [CrossRef] [PubMed]
Boulton M Dontsov A Jarvis-Evans J Ostrovsky M Svistunenko D . Lipofuscin is a photoinducible free radical generator. J Photochem Photobiol B. 1993;19:201–204. [CrossRef] [PubMed]
Liu J Itagaki Y Ben-Shabat S Nakanishi K Sparrow JR . The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane. J Biol Chem. 2000;275:29354–29360. [CrossRef] [PubMed]
Dorey CK Wu G Ebenstein D Garsd A Weiter JJ . Cell loss in the aging retina. Relationship to lipofuscin accumulation and macular degeneration. Invest Ophthalmol Vis Sci. 1989;30:1691–1699. [PubMed]
Kennedy CJ Rakoczy PE Constable IJ . Lipofuscin of the retinal pigment epithelium: a review. Eye. 1995;9:763–771. [CrossRef] [PubMed]
Bird A . Age-related macular disease. Br J Ophthalmol. 1996;80:2–3. [CrossRef] [PubMed]
Young RW . Pathophysiology of age-related macular degeneration. Surv Ophthalmol. 1987;31:291–306. [CrossRef] [PubMed]
Mullins RF Russell SR Anderson DH Hageman GS . Drusen associated with aging and age-related macular degeneration contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease. FASEB J. 2000;14:835–846. [PubMed]
Ambati J Anand A Fernandez S . An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice. Nat Med. 2003;9:1390–1397. [CrossRef] [PubMed]
Ferris FL3rd Fine SL Hyman L . Age-related macular degeneration and blindness due to neovascular maculopathy. Arch Ophthalmol. 1984;102:1640–1642. [CrossRef] [PubMed]
Hageman GS Luthert PJ Victor Chong NH Johnson LV Anderson DH Mullins RF . An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch's membrane interface in aging in age-related macular degeneration. Prog Retin Eye Res. 2001;20:705–732. [CrossRef] [PubMed]
Johnson LV Leitner WP Staples MK Anderson DH . Complement activation and inflammatory processes in Drusen formation and age related macular degeneration. Exp Eye Res. 2001;73:887–896. [CrossRef] [PubMed]
Bird AC Bressler NM Bressler SB . An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group. Surv Ophthalmol. 1995;39:367–374. [CrossRef] [PubMed]
Bressler SB Maguire MG Bressler NM Fine SL . Relationship of drusen and abnormalities of the retinal pigment epithelium to the prognosis of neovascular macular degeneration. The Macular Photocoagulation Study Group. Arch Ophthalmol. 1990;108:1442–1447. [CrossRef] [PubMed]
Haines JL Hauser MA Schmidt S . Complement factor H variant increases the risk of age-related macular degeneration. Science. 2005;308:419–421. [CrossRef] [PubMed]
Hageman GS Anderson DH Johnson LV . A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci U S A. 2005;102:7227–7232. [CrossRef] [PubMed]
Edwards AO Ritter R3rd Abel KJ Manning A Panhuysen C Farrer LA . Complement factor H polymorphism and age-related macular degeneration. Science. 2005;308:421–424. [CrossRef] [PubMed]
Morgan BP . Complement membrane attack on nucleated cells: resistance, recovery, and non-lethal effects. Biochem J. 1989;264:1–14. [PubMed]
Kilgore KS Flory CM Miller BF Evans VM Warren JS . The membrane attack complex of complement induces IL-8 and monocyte chemoattractant protein-1 secretion from human umbilical vein endothelial cells. Am J Pathol. 1996;149:953–961. [PubMed]
Anderson DH Mullins RF Hageman GS Johnson LV . A role for local inflammation in the formation of drusen in the aging eye. Am J Ophthalmol. 2002;134:411–431. [CrossRef] [PubMed]
Lommatzsch A Hermans P Müller KD Bornfeld N Bird AC Pauleikhoff D . Are low inflammatory reactions involved in exudative age-related macular degeneration? Morphological and immunhistochemical analysis of AMD associated with basal deposits. Graefes Arch Clin Exp Ophthalmol. 2007;246:803–810. [CrossRef]
Izumi-Nagai K Nagai N Ozawa Y . Interleukin-6 receptor-mediated activation of signal transducer and activator of transcription-3 (STAT3) promotes choroidal neovascularization. Am J Pathol. 2007;170:2149–2158. [CrossRef] [PubMed]
Roh MI Kim HS Song JH Lim JB Koh HJ Kwon OW . Concentration of cytokines in the aqueous humor of patients with naïve, recurrent and regressed CNV associated with amd after bevacizumab treatment. Retina. 2009;29:523–529. [CrossRef] [PubMed]
Jonas JB Tao Y Neumaier M Findeisen P . Monocyte chemoattractant protein 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in exudative age-related macular degeneration. Arch Ophthalmol. 2010;128:1281–1286. [CrossRef] [PubMed]
Ferrara N Gerber HP LeCouter J . The biology of VEGF and its receptors. Nat Med. 2003;9:669–676. [CrossRef] [PubMed]
Frank RN Amin RH Eliott D Puklin JE Abrams GW . Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes. Am J Ophthalmol. 1996;122:393–403. [CrossRef] [PubMed]
Kvanta A Algvere PV Berglin A Seregard S . Subfoveal fibrovascular membrane in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci. 1996;37:1929–1934. [PubMed]
Dumont P Burton M Chen QM . Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast. Free Radic Biol Med. 2000;28:361–373. [CrossRef] [PubMed]
Toussaint O Medrano EE von Zglinicki T . Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. Exp Gerontol. 2000;35:927–945. [CrossRef] [PubMed]
Molday RS Molday LL . Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method. J Cell Biol. 1987;105:2589–2601. [CrossRef] [PubMed]
Higgins GT Wang JH Dockery P Cleary PE Redmond HP . Induction of angiogenic cytokine expression in cultured RPE by ingestion of oxidizes photoreceptor outer segments. Invest Ophthalmol Vis Sci. 2003;44:1775–1782. [CrossRef] [PubMed]
Chen W Kang J Xia J . p53-related apoptosis resistance and tumor suppression activity in UVB-induced premature senescent human skin fibroblasts. Int J Mol Med. 2008;21:645–653. [PubMed]
Omwancha J Anway MD Brown TR . Differential age-associated regulation of clusterin expression in prostate lobes of brown Norway rats. Prostate. 2009;69:115–125. [CrossRef] [PubMed]
Sahlin S Hed J Rundquist I . Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay. J Immunol Methods. 1983;60:115–124. [CrossRef] [PubMed]
Reddy SM Hsiao KH Abernethy VE . Phagocytosis of apoptotic cells by macrophages induces novel signalling events leading to cytokine-independent survival and inhibition of proliferation: activation of Akt and inhibition of extracellular signal-regulated kinases 1 and 2. J Immunol. 2002;169:702–713. [CrossRef] [PubMed]
Garcia-Diaz JF Essig A . Capacitative transients in voltage-clamped epithelia. Biophys J. 1985;48:519–523. [CrossRef] [PubMed]
Abe T Sugano E Saigo Y Tamai M . Interleukin-1beta and barrier function of retinal pigment epithelial cells (ARPE-19): aberrant expression of junctional complex molecules. Invest Ophthalmol Vis Sci. 2003;44:4097–4104. [CrossRef] [PubMed]
Wing GL Blanchard GC Weiter JJ . The topography and age relationship of lipofuscin concentration in the retinal pigment epithelium. Invest Ophthalmol Vis Sci. 1978;17:601–607. [PubMed]
Yu AL Fuchshofer R Kook D Kampik A Bloemendal H Welge-Lüssen U . Subtoxic oxidative stress induces senescence in retinal pigment epithelial cells via TGF-ß release. Invest Ophthalmol Vis Sci. 2009;50:926–935. [CrossRef] [PubMed]
Crabb JW Miyagi M Gu X . Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc Natl Acad Sci U S A. 2002;99:14682–14687. [CrossRef] [PubMed]
Lee C Sensibar JA . Proteins of the rat prostate II: synthesis of new proteins in the ventral lobe during castration induced regression. J Urol. 1987;138:903–908. [PubMed]
Dumont P Chainiaux F Eliaers F . Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide. Cell Stress Chaperones. 2002;7:23–35. [CrossRef] [PubMed]
Burke JM Skumatz CM . Autofluorescent inclusions in long-term postconfluent cultures of retinal pigment epithelium. Invest Ophthalmol Vis Sci. 1998;39:1478–1486. [PubMed]
Eldred CE Laskey MR . Retinal age pigments generated by selfabsorbing lysosomotropic detergents. Nature. 1993;361:724–726. [CrossRef] [PubMed]
Delori FC Goger DG Dorey CK . Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects. Invest Ophthalmol Vis Sci. 2001;42:1855–1866. [PubMed]
Holz FG Bellmann C Margaritidis M Schütt F Völcker HE . Patterns of increased in vivo fundus autofluorescence in the junctional zone of geographic atrophy of the retinal pigment epithelium associated with age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol. 1999;237:145–152. [CrossRef] [PubMed]
Feeney-Burns L Hilderbrand ES Eldridge S . Aging human RPE: morphometric analysis of macular, equatorial, and peripheral cells. Invest Ophthalmol Vis Sci. 1984;25:195–200. [PubMed]
Brunk UT Terman A . The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis. Eur J Biochem. 2002;269:1996–2002. [CrossRef] [PubMed]
Cuervo AM Hu W Lim B Dice JF . IkappaB is a substrate for a selective pathway of lysosomal proteolysis. Mol Biol Cell. 1998;9:1995–2010. [CrossRef] [PubMed]
Szweda PA Camouse M Lundberg KC Oberley TD Szweda LI . Aging, lipofuscin formation, and free radical-mediated inhibition of cellular proteolytic systems. Ageing Res Rev. 2003;2:383–405. [CrossRef] [PubMed]
Holz FG Schütt F Kopitz J Völcher HE . Introduction of the lipofuscin fluorophore A2E into the lysosomal compartment of human retinal pigment epithelial cells by coupling to LDL particles. An in vitro model of retinal pigment epithelium cell aging. Ophthalmologe. 1999;96:781–785. [CrossRef] [PubMed]
Schütt F Davies S Kopitz J Holz FG Boulton ME . Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin. Invest Ophthalmol Vis Sci. 2000;41:2303–2308. [PubMed]
Sparrow JR Parish CA Hashimoto M Nakanishi K . A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture. Invest Ophthalmol Vis Sci. 1999;40:1988–1995.
Thurman JM Renner B Kunchithapautham K . Oxidative stress renders retinal pigment epithelial cells susceptible to complement-mediated injury. J Biol Chem. 2009;284:16939–16947. [CrossRef] [PubMed]
Lueck K Wasmuth S Williams J . Sub-lytic C5b-9 induces functional changes in retinal pigment epithelial cells consistent with age-related macular degeneration. Eye. 2011;25:1074–1082. [CrossRef] [PubMed]
Holtkamp GM Van Rossem M de Vos AF Willekens B Peek R Kijlstra A . Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells. Clin Exp Immunol. 1998;112:34–43. [CrossRef] [PubMed]
Strande JL Phillips SA . Thrombin increases inflammatory cytokine and angiogenic growth factor secretion in human adipose cells in vitro. J Inflamm. 2009;6:4. [CrossRef]
Fukuoka Y Strainic M Medof ME . Differential cytokine expression of human retinal pigment epithelial cells in response to stimulation by C5a. Clin Exp Immunol. 2003;131:248–253. [CrossRef] [PubMed]
Figure 1.
 
Increased expression of senescence-associated genes of UV-POS treated RPE cells compared with untreated and POS-treated cells analyzed by RT-PCR (A, B). Data are normalized mean and standard deviations from four independent experiments. * P < 0.05.
Figure 1.
 
Increased expression of senescence-associated genes of UV-POS treated RPE cells compared with untreated and POS-treated cells analyzed by RT-PCR (A, B). Data are normalized mean and standard deviations from four independent experiments. * P < 0.05.
Figure 2.
 
Elevated total and intracellular AF of UV-POS treated RPE cells compared with untreated and POS treated cells. (A) The total AF was determined photometrically at a wavelength of 490 nm. Presented is the mean out of 16 separate measurements. (B) Representative histogram of the total AF from four different experiments measured by flow cytometry at a wavelength of 488 nm. (C) Histogram of the intracellular AF, representing internalized POS and UV-POS, analyzed by flow cytometry. Untreated, gray histogram; POS, open gray histogram; UV-POS, open black histogram.
Figure 2.
 
Elevated total and intracellular AF of UV-POS treated RPE cells compared with untreated and POS treated cells. (A) The total AF was determined photometrically at a wavelength of 490 nm. Presented is the mean out of 16 separate measurements. (B) Representative histogram of the total AF from four different experiments measured by flow cytometry at a wavelength of 488 nm. (C) Histogram of the intracellular AF, representing internalized POS and UV-POS, analyzed by flow cytometry. Untreated, gray histogram; POS, open gray histogram; UV-POS, open black histogram.
Figure 3.
 
Increased accumulation of UV-POS in RPE cells. Representative Hoechst staining (blue) of paraffin sections prepared of RPE cells grown on polycarbonate cell-culture inserts (Transwell; Corning), incubated apically with labeled (red) (A) POS and (B) UV-POS. (C) Representative Western blot analysis results for rhodopsin using cell lysates of POS and UV-POS treated cells demonstrating a decreased degradation of UV-POS. (D) Densitometric analysis of rhodopsin Western blot bands (n = 5) normalized to α-tubulin. *P < 0.05.
Figure 3.
 
Increased accumulation of UV-POS in RPE cells. Representative Hoechst staining (blue) of paraffin sections prepared of RPE cells grown on polycarbonate cell-culture inserts (Transwell; Corning), incubated apically with labeled (red) (A) POS and (B) UV-POS. (C) Representative Western blot analysis results for rhodopsin using cell lysates of POS and UV-POS treated cells demonstrating a decreased degradation of UV-POS. (D) Densitometric analysis of rhodopsin Western blot bands (n = 5) normalized to α-tubulin. *P < 0.05.
Figure 4.
 
Decreased neutral red uptake through UV-POS treated RPE cells compared with untreated or POS-treated cells. Illustrated are mean and standard deviations from eight independent experiments. *P < 0.05.
Figure 4.
 
Decreased neutral red uptake through UV-POS treated RPE cells compared with untreated or POS-treated cells. Illustrated are mean and standard deviations from eight independent experiments. *P < 0.05.
Figure 5.
 
Verification of a dense cell monolayer. (A) TER measurements and (B) permeability assay on RPE cells grown on polycarbonate cell culture inserts (Transwell; Corning) or inserts without cells as a control. (A) The TER was measured once a week. Mean and standard deviations were evaluated from triplicate measurements from 10 inserts. (B) Apical to basal movement of sodium fluorescein was determined at different time points. Presented are mean and SD of representative duplicate tests.
Figure 5.
 
Verification of a dense cell monolayer. (A) TER measurements and (B) permeability assay on RPE cells grown on polycarbonate cell culture inserts (Transwell; Corning) or inserts without cells as a control. (A) The TER was measured once a week. Mean and standard deviations were evaluated from triplicate measurements from 10 inserts. (B) Apical to basal movement of sodium fluorescein was determined at different time points. Presented are mean and SD of representative duplicate tests.
Figure 6.
 
Decreased TER of ARPE-19 cells grown on polycarbonate cell culture inserts (Transwell; Corning) after incubation with UV-POS and HCS compared with untreated, POS, or C7-deficient HCS treated cells. Presented are mean and SD from four independent experiments. *P < 0.05.
Figure 6.
 
Decreased TER of ARPE-19 cells grown on polycarbonate cell culture inserts (Transwell; Corning) after incubation with UV-POS and HCS compared with untreated, POS, or C7-deficient HCS treated cells. Presented are mean and SD from four independent experiments. *P < 0.05.
Figure 7.
 
IL-6 (A), IL-8 (B), MCP-1 (C), and VEGF (D) secretion toward the basal side by untreated, POS, or UV-POS treated RPE cells, as well as in addition with C7-deficient HCS and HCS. Shown are mean and SD from four separate experiments collected by ELISA. *P < 0.05.
Figure 7.
 
IL-6 (A), IL-8 (B), MCP-1 (C), and VEGF (D) secretion toward the basal side by untreated, POS, or UV-POS treated RPE cells, as well as in addition with C7-deficient HCS and HCS. Shown are mean and SD from four separate experiments collected by ELISA. *P < 0.05.
Table 1.
 
Primer Sequences and PCR Conditions
Table 1.
 
Primer Sequences and PCR Conditions
Target Sequence from 5′ to 3′ bp Initial Step Denaturation, Annealing, Elongation Number of Cycles Final Elongation
Apo J F gaa atg aag ctg aag gct ttc ccg 286 5′ at 95°C 45 s at 94°C, 45 s at 57°C, 1′ at 72°C 24 10′ at 72°C
B gga act gta aag ctg ggc tat gga
SM22 F tga agg tgc ccg aga acc ca 367 30 s at 94°C, 40 s at 57°C, 30 s at 72°C 35
B atc tgc cga ggt cgt ccg tag c
Fibronectin F tgc caa cct tta cag acc ta 492 30 s at 94°C, 40 s at 57°C, 30 s at 72°C 35
B ctc atc tcc ctc ctc act ca
Osteonectin F gca gag gaa acc gaa gag ga 207 30 s at 94°C, 40 s at 57°C, 30 s at 72°C 35
B ggc aaa gaa gtg gca gga ag
GAPDH F atg aca tca aga agg tgg tg 177 30 s at 94°C, 30 s at 55°C, 1′ at 72°C 25
B cat acc agg aaa tga gct tg
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