Induction of Amyloid β Accumulation by ER Calcium Disruption and Resultant Upregulation of Angiogenic Factors in ARPE19 Cells

Abstract

purpose. To investigate the intracellular mechanisms that induce amyloid β (Aβ) accumulation and angiogenesis in the human retinal pigment epithelial cell line ARPE19.

methods. The authors used two endoplasmic reticulum (ER) stress-inducing reagents, thapsigargin (TG), which inhibits the sarcoplasmic/endoplasmic calcium (Ca)²⁺-ATPase, and tunicamycin (TM), which inhibits N-linked glycosylation. The expression pattern of Aβ-precursor protein (APP) splice variants was investigated by reverse transcription (RT)-PCR. Cellular expressions of both a series of Aβ metabolism-related factors and angiogenic factors were evaluated by real-time RT-PCR and Western blot (VEGF). Expression of caspase-4 was examined by real-time RT-PCR and Western blot to evaluate the effect of the ER stressor. Intracellular Ca elevation by TG was evaluated by Ca²⁺ imaging experiments. Dimethyl sulfoxide and staurosporine were used as a nonreagent control and as an apoptosis-inducing reagent through mitochondria not ER, respectively.

results. TG-treated ARPE19 cells increased the mRNA expression of Aβ production-inducing APP splice variants and reduced that of neprilysin, a catabolic enzyme for Aβ. TG-treated ARPE19 cells produced increases in VEGF, TNF-α, TACE mRNA, and VEGF protein expressions and a decrease in PEDF mRNA expression. TG-treated ARPE19 cells induced the expression of active more than TM-treated casepase-4. The intracellular Ca concentration was elevated in only TG-treated ARPE19 cells.

conclusions. TG-treated ARPE19 cells showed both Aβ accumulation-inducible and angiogenic factor mRNA expression patterns. This study suggests the possibility that ER stress through ER calcium disruption may induce the expression not only of Aβ deposit-promoting factors but also angiogenic factors in the retinal pigment epithelium.

Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. ¹ Although our understanding of molecular events presaging AMD has grown in the past decade, the pathogenesis of AMD remains poorly understood. AMD is classified as dry or nonexudative AMD and as wet or exudative AMD. The most severe complication in wet or exudative AMD is the development of choroidal neovascularization (CNV), which originates in choroidal blood vessels that grow through Bruch membrane into the sub-retinal pigment epithelium (RPE) or the subretinal space, or both. The clinical hallmark of AMD is the appearance of drusen, ² localized deposits lying between the basement membrane of the RPE and Bruch membrane. 

Recently, new evidence has indicated that, in AMD, substructural elements within drusen contain amyloid β (Aβ), ^{3 4 5} which is a major component of senile plaques and cerebrovascular deposits found in the brains of patients with Alzheimer disease (AD). Aβ accumulation has been reported to increase the expression of an angiogenic growth factor, vascular endothelial growth factor (VEGF), which plays an important role in conditions that involve ocular angiogenesis, including CNV, ^{6 7 8 9} and to decrease the expression of the potent antiangiogenic factor ^{10 11} pigment epithelium-derived factor (PEDF), secreted by retinal pigment epithelial cells. ¹² These results suggest that Aβ is related to the pathogenesis of AMD. However, the mechanisms that induce the accumulation of Aβ in the RPE of AMD patients have not been determined. 

Aβ is known to be a physiological peptide, the steady state level of which is maintained by a metabolic balance between synthesis and degradation, and is constitutively secreted from cells. ^{13 14} In the amyloidogenic pathway, Aβ is produced by the sequential proteolytic processing of Aβ-precursor protein (APP) by the β site APP cleaving enzyme (BACE) and a presenilin complex. ^{15 16 17} Under physiological conditions, Aβ is degraded by peptidases, such as neprilysin ¹⁸ and endothelin-converting enzyme (ECE), ¹⁹ immediately after production. ²⁰  

Endoplasmic reticulum (ER) stress, which leads to the accumulation of unfolded protein, results in ER dysfunction and subsequent cell death. ²¹ Neuro 2a cells expressing presenilin2-splice variants, which is expressed in human brains in sporadic AD, or the dominant-negative form of Ire1 are vulnerable to ER stress and to increased Aβ production. ²² Therefore, ER stress plays an important role in Aβ accumulation. We used two ER-stress inducers, thapsigargin (TG) and tunicamycin (TM), in this study. TG, a highly lipophilic sesquiterpene lactone, is broadly used as a selective inhibitor of the sarcoplasmic reticulum calcium-ATPase, which pumps Ca²⁺ from the cytosol into the lumen of ER in mammalian cells. TG-mediated irreversible inhibition of ER Ca²⁺ ATPases can also cause the induction of Ca²⁺ leakage from the ER to the cytoplasm, further facilitating the depletion of Ca²⁺ within the ER, and can result in increases in cytoplasmic Ca²⁺ levels. ²³ Long-term elevations of intracellular Ca induced ER stress from abnormal accumulations of folding protein. ^{24 25} TM is the glucosamine-containing nucleoside antibiotic generated by Streptomyces and an inhibitor of N-linked glycosylation and by the formation of N-glycosidic protein-carbohydrate linkages. ²⁶ It specifically inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues of glycoproteins, ²⁷ accumulates the unfolded proteins, and induces ER stress. ^{25 28} To determine the intracellular mechanisms that induce Aβ accumulation in RPE, we treated human retinal pigment epithelial cells, ARPE19 cells, with various ER-stress inducers, such as TM and TG, and investigated the responses of the Aβ accumulation-inducible event. 

Materials and Methods

Cell Culture

ARPE19 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in Dulbecco modified Eagle medium/F-12 human amniotic membrane nutrient mixture (DMEM/F-12; Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (FBS; Sigma-Aldrich) in a humidified incubator at 37°C in an atmosphere of 5% CO₂. The medium was changed every 3 days. 

Preparation of Reagents

TG (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at a concentration of 0.5 mM to produce stock solutions. These solutions were diluted to 1:500 with medium to obtain 1 μM TG-containing culture medium. To evaluate the effect of DMSO, medium containing only DMSO (1:500 of total volume) was also prepared for each study. In comparison with TG, 1 μM TM (Sigma-Aldrich) and 0.1 μM staurosporine (STS; Sigma-Aldrich), the bacterial alkaloid that did not induce apoptosis through the ER, ²⁹ were used for each study. Both TG and TM were reported to upregulate VEGF mRNA expression in ARPE19 cells. ³⁰ The concentrations of TG and TM used to treat ARPE19 cells were the ones that induced VEGF mRNA expression maximally in all experiments in this study. The concentration of STS was selected based on a previous paper. ³¹ Every reagent was exposed to the ARPE19 cells for 24 hours. 

RNA Isolation and cDNA Preparation

Total RNA from ARPE19 cells with exposure to each reagent was isolated (RNA Easy Kit; Qiagen, Tokyo Japan) and quantified by ultraviolet spectrometry at 260 nm. One microgram of RNA was transcribed to cDNA using reverse transcription reagents (Superscript III; Invitrogen, Carlsbad, CA). 

Real-Time RT-PCR

Real-time RT-PCR was performed on a thermocycler (7900HT Sequence Detection Systems; Applied Biosystems, Foster, CA) with nuclear stain (SYBR Green; Applied Biosystems) reagents according to the manufacturer’s instructions. Amplification of PCR products was measured by fluorescence associated with binding of double-stranded DNA to the SYBR green dye in the reaction mixture. Sequences of the primers used in this study are listed in Table 1 . After an initial denaturation step of 50°C for 2 minutes and 95°C for 10 minutes, PCR involved 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute. Quantification of each PCR product was expressed relative to GAPDH. 

RT-PCR

For semiquantitative experiments, each PCR amplification was tested to reach half the saturation curve, and an aliquot of cDNA libraries was amplified by PCR with specific oligonucleotides for APP isoforms APP770, APP751, and APP695 (605, 586, and 418 bp, respectively, of PCR product). APP primers were designed to flank the alternatively spliced exons (exons 7 and 8) to detect the expression of the three major APP isoforms found in the brain. Oligonucleotide sequences used were as follows: sense, 5′-aga gag aac cac cag cat tgc c-3′ (992–1013 bp); antisense, 5′-ggt cat tga gca tgg ctt cca c-3′ (1575–1596). These sequences were designed from the human APP cDNA sequences corresponding to GenBank accession number NM_000484. Amplification was performed in a thermocycler (PCR System 9700; Applied Biosystems). Conditions of amplification were 30 seconds at 95°C, 30 seconds at 57°C, and 1 minute at 72°C for 28 cycles to detect APP770 and APP751 or for 33 cycles to detect APP695. Finally, 5% polyacrylamide gels were stained by ethidium bromide and acquired with a CCD camera. mRNA levels were quantified using NIH image and normalized to the GAPDH levels. 

Western Blot

Cells were homogenized in lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 150 mM NaCl, 0.5% NP-40) and were stored in sample buffer (50 mM TBS containing 2% sodium dodecyl sulfate [SDS], 6% β-mercaptoethanol, 10% glycerol) at −20°C until use. Quantification of the protein contents was made by the Bradford method. 

Protein samples (15 μg) were separated on SDS-PAGE (12% acrylamide) and were transferred to polyvinylidene difluoride filters (Millipore, Bedford, MA). The filters were blocked in 0.1 M PBS containing 5% skim milk and 0.05% Tween 20 for 1 hour at room temperature and were incubated overnight at 4°C with a monoclonal mouse anti–caspase-4 (1:1000; MBL, Nagano Japan) and a polyclonal rabbit anti-VEGF (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) primary antibody. After five washes in 0.1 M PBS containing 0.05% Tween 20, the filters were incubated for 1 hour at room temperature with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:1000; Cell Signaling, Beverly, MA) and an anti–rabbit IgG (1:1000; Cell Signaling) secondary antibody, washed, visualized in ECL solution (Amersham Biosciences, Arlington Heights, IL) for 10 minutes, and exposed onto film (X-Omat; Fuji, Kanagawa, Japan) for 7 to 10 minutes. Finally, the filters were incubated in a stripping buffer (2% SDS, 0.7% β-mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8) for 30 minutes at 65°C and were reprobed with a monoclonal mouse anti–β-actin antibody (1:3000; Chemicon, Temecula, CA) as loading controls. Our Western blot bands showed the same band sizes as indicated in the antibody information sheets. Protein levels were quantified by densitometry and normalized to the β-actin levels. 

Data and Statistical Analyses

Statistical analysis of mRNA and protein expression levels for each reagent treatment was performed using the Student’s t-test in comparisons with DMSO treatment (control). Data were expressed as mean ± SE (n = 3). 

Intracellular Ca²⁺ Measurement

The intracellular Ca²⁺ concentration in retinal pigment epithelial cells was monitored using the Ca²⁺ indicator dye (Fluo-4; Invitrogen). After 3 days in vitro, cells were incubated for 20 minutes with the cell-permeant acetomethyl ester form of 5 mM Ca²⁺ indicator dye (Fluo-4; Invitrogen) diluted in culture medium containing 0.01% pluonic acid F-127 at 37°C. The cells were subsequently washed three times with culture medium, and the dye was allowed to deesterify for an additional 30 minutes in the CO₂ incubator. Ca²⁺ imaging experiments were performed using a live cell imaging microscope system (BioStation IM; Nikon, Tokyo, Japan) with a 20× objective. Fluorescent images were acquired every 20 seconds. Fluo-4 fluorescence was produced by excitation from a 130-W mercury-vapor lamp and an appropriate filter set (B-2A [Nikon]; excitation, 450–490 nm; emission, 520–560 nm; dichroic, 510 nm). All drugs were applied by bath application after 5-minute control observation. Data analysis was performed as follows: off-line analysis of the images was performed using commercial software (Aquacosmos; Hamamatsu Photonics, Hamamatsu, Japan). Fluorescence signal was quantified by measuring the average pixel intensity within cell bodies. Changes in fluorescence intensity of each cell were normalized to its baseline fluorescence intensity. 

Results

Induction of Aβ Production-Inducible APP Splice Variants mRNA Expression in ER-Stress–Treated ARPE19 Cells

In the amyloidogenic pathway, the cleavage of APP by the β-secretase and presenilin complex results in Aβ production. APP has several splice variants. Exons 7, 8, and 15 of the APP gene can be alternatively spliced to produce multiple isoforms. In the brain, the major isoform transcripts result from the splicing of exons 7 and 8, which gives rise to APP695, APP751, and APP770. ²⁰ Both APP770 and APP751 contain a serine protease inhibitory domain encoded by exon 7 called the Kunitz protease inhibitory domain (KPI). KPI-APP has been shown to be more amyloidogenic than KPI-deficient APP. ³²  

In the ARPE19 cells, the exposure of TG, which induces a depletion of intracellular Ca stores, slightly increased the expression of KPI-APP, such as APP751 and APP770 (Fig. 1A) . TM, which inhibits N-linked glycosylation, induced an increase of KPI-APP expression in ARPE19 cells (Fig. 1A) . STS, which is widely used as an inducer of non–ER stress-induced apoptosis, did not induce the expression of KPI-APP (Fig. 1A) . Real-time RT-PCR showed no change in the total APP mRNA expression of any of the reagent treatments (Fig. 1B) . These results indicate that ER stress induces mRNA expression of APP splice variants, which accelerate Aβ accumulation, in ARPE19 cells. 

Decrease of Aβ Metabolism-Related Factors mRNA Expression in TG-Treated ARPE19 Cells

To find the relationship between ER stress–inducing factors and cellular mRNA expression of a series of Aβ metabolism–related factors (such as neprilysin, ECE, and BACE) in ARPE19 cells, we performed real-time RT-PCR. 

TG-treated ARPE19 cells showed a remarkable decrease in mRNA expression of neprilysin, which highly degrades Aβ ^{33 34} (Fig. 2A) . The mRNA expression of ECE, which degrades Aβ, and of BACE, which cleaves APP at the β site, was not quantitatively changed by TG (Figs. 2B 2C) . The expression of all investigated Aβ metabolism-related genes were not affected by TM or STS treatment (Fig. 2) . Because there was a remarkable decrease of neprilysin mRNA observed in only TG-treated cells, it is suggested that the degradation of Aβ is depressed by a decrease in the expression of neprilysin in ARPE19 cells. 

Induction of Angiogenic Factors mRNA Expression in ER-Stress–Treated ARPE19 Cells

It has been reported that exposure to Aβ induced the mRNA expression of VEGF and a reduction of PEDF in ARPE19 cells. ¹² We investigated the cellular mRNA expression change of VEGF and PEDF produced by TG and TM treatment in ARPE19 cells. In addition, real-time RT-PCR was performed for tumor necrosis factor alpha (TNF-α) and TNF-α converting enzyme (TACE). TNF-α, a macrophage/monocyte-derived polypeptide, stimulates VEGF production in human glioma cells and retinal pigment epithelial cells, ^{35 36} and TACE is involved the generation of soluble TNF-α from membrane-bound TNF-α and promotes angiogenesis. ^{37 38}  

TG and TM exclusively upregulated VEGF mRNA expression, as reported previously, ³⁰ as did STS (Fig. 3A) . Real-time RT-PCR showed that TG and STS exclusively downregulated PEDF mRNA expression, and TM did not change (Fig. 3B) . TG and TM also upregulated the mRNA expression of TNF-α and TACE (Figs. 3C 3D) . STS upregulated the mRNA expression of TNF-α but did not change that of TACE (Figs. 3C 3D) . To confirm the increase of VEGF expression at the protein level, we performed Western blot analysis for VEGF and showed that TG and TM also upregulated VEGF protein expression (Fig. 3E) . These results suggest that the change in expression of these angiogenic factors might be the result of a general apoptotic event. 

Induction of Caspase-4 Expression in TG-Treated ARPE19 Cells

Caspase-4 is primarily activated in ER stress-induced apoptosis. ³⁹ Real-time RT-PCR and Western blot showed that the expression of caspase-4 mRNA and the active form of its protein were upregulated by TG treatment (Fig. 4) . Although expression of the active form of caspase-4 protein was also upregulated by TM treatment, it was much lower than that of TG-treated ARPE19 cells (Fig. 4) . STS-treated ARPE19 cells showed no change in expression of caspase-4 (Fig. 4) . 

Induction of Intracellular Ca Elevation in TG-Treated ARPE19 Cells

TG can inhibit the sarcoplasmic reticulum calcium-ATPase and can elevate intracellular Ca concentration. ²³ We performed Ca imaging (Fluo-4 and Fura-2/AM; Invitrogen) to investigate whether the intracellular Ca concentration was actually increased in TG-treated ARPE19 cells. TG-treated ARPE19 cells showed a remarkable increase in intracellular Ca concentration (Fig. 5and Supplementary Fig. S1). TM- and STS-treated ARPE19 cells showed no change in intracellular Ca concentration (Fig. 5and Supplementary Fig. S1). 

Discussion

Recently, it has been reported that Aβ accumulates in the RPE of patients with AMD, ^{3 4 5} which is the most common cause of vision loss in the development of CNV. Aβ, known as the hallmark of AD, is routinely produced in the normal brain and is readily degraded by catabolic enzymes. However, facilitation of Aβ production is caused by mutation of the gene responsible for AD. Aβ then accumulates as the insoluble senile plaques in the brain. ER stress is considered one of the causative factors for the accumulation of Aβ in AD. ⁴⁰ To examine the intracellular machinery for Aβ accumulation in the RPE of patients with AMD, we treated human retinal pigment epithelial cells, ARPE19 cells, with the ER-stress inducer and investigated whether Aβ accumulation was inducible. Our results suggested that Aβ accumulation-inducible factors were induced by ER stress in retinal pigment epithelial cells as well as in neural cells, and angiogenic factors were additively increased in retinal pigment epithelial cells by ER stress under the same conditions. 

We showed that ER stress treatment induced an increase in mRNA expression of Aβ production-inducible APP splice variants; only TG treatment further induced a decrease in the mRNA expression of neprilysin in ARPE19 cells (Figs. 1 2) . It has been reported that KPI-APP, which increases at the mRNA level in the brains of persons with AD, ⁴¹ is more amyloidogenic than KPI-deficient APP. ³² Therefore, it is thought that the increase in mRNA expression of KPI-APP induced the production of Aβ. On the other hand, neprilysin knockout mice have been reported to display an approximately 50% increase in the levels of Aβ40 and Aβ42. ¹⁴ Based on these facts, we surmised that the increased Aβ production and the decreased Aβ degradation induced specifically by TG treatment in ARPE19 cells might be attributed to the Aβ accumulation in ARPE19 cells. 

We also confirmed that the upregulation of TNF-α, TACE, and VEGF and the downregulation of PEDF were induced by TG treatment in ARPE19 cells at the mRNA level (Figs. 3A 3B 3C 3D) . Moreover, VEGF protein expression was upregulated by TG treatment (Fig. 3E) , suggesting that TG treatment induced the expression not only of Aβ accumulation-promoting factor expression but also of angiogenic factors in ARPE19 cells. 

Because transcription factor 4, activated by ER stress, induced VEGF, ⁴² ER stress has been considered to induce not only Aβ accumulation but also angiogenesis. However, our results showed a pronounced difference in the responses of Aβ accumulation-promoting factors and angiogenic factors between TG and TM, likely because of two reasons. First, there was a difference in the extent of ER stress-induced apoptosis. Caspase-4, which is primarily activated in ER stress-induced apoptosis, ³⁹ was shown to be activated in TG-treated ARPE19 cells more than in TM-treated ARPE19 cells (Fig. 4) . Second, we conjectured that the difference in results was attributed to the mechanism of action of each ER stress inducer. TG-induced ER stress was caused by the depletion of intracellular Ca stores, ²⁴ whereas TM-induced ER stress was caused by an inhibition of N-linked glycosylation. ²⁸ Differing from both TM- and STS-treated ARPE19 cells, we showed TG-treated ARPE19 cell exhibited striking increases in intracellular Ca concentration. It has been reported that insulin-like growth factor-1 stimulates increased VEGF secretion through the induction of the second messenger Ca in ARPE19 cells. ⁴³ The elevation of intracellular Ca levels was also reported to increase Aβ peptide production through the activation of a protease, which requires Ca, in the cultured cells. ⁴⁴ Based on these facts, intracellular increases of Ca by TG treatment may induce Aβ accumulation and angiogenic factors in ARPE19 cells more effectively than ER stress induced by TM. 

It has long been known that the elevation of intracellular free calcium by TG has cytotoxic consequence in many cells (adipocytes, ⁴⁵ T lymphocytes, ⁴⁶ parotid acinar cells, ⁴⁷ and peritoneal macrophages ⁴⁸ ) and cell lines (hepatocytes, ⁴⁹ HeLa cells, ⁵⁰ and NG115–401L cells ²⁴ ), including ARPE19 cells. ⁵¹ TM was used mainly for analysis in N-glycosylation (except that TM was used as an ER-stress inducer), whereas TG is involved in various cellular functions, including platelet activation, ⁵² inflammation, ⁵³ skin irritation, ⁵⁴ protein synthesis inhibition in human hepatoma cells, ⁵⁵ vascular contractility, ⁵⁶ and tumor promotion. ⁵⁵ Moreover, it has been reported that TG induced Bax activation and was involved in mitochondrial caspase-dependent death. ⁵⁷ It was often said that the low concentrations (0.5–2 μM) of TG increased the intracellular Ca and high concentration (1–10 μM) of TG-induced apoptosis (refer to Sigma data sheet). In addition, it has been reported that long-term (48-hour) exposure of TG led to recovery from upregulation of the intracellular Ca concentration ⁵⁸ and the induction of cell death. ⁴⁹ In our study, TG was exposed at 1 μM for 24 hours because we wanted to determine the change of various factors in the viable retinal pigment epithelial cells at early stages of Aβ accumulation. Our results were consistent with those of a previous report ⁵¹ in the elevation of the intracellular Ca concentration (Fig. 5) . However, the change of BAX expression was not induced, probably because of the lower concentration and shorter incubation time of TG (data not shown). Therefore, TG would induce Aβ accumulation and angiogenic responses in retinal pigment epithelial cells mainly through induction of the intracellular Ca concentration in this study. Further studies would be needed to determine the responses under various conditions of TG incubation. 

It has been reported that neprilysin inhibits angiogenesis through the proteolysis of fibroblast growth factor-2 (FGF-2), ⁵⁹ and neprilysin knockout mice display an upregulation of VEGF and a downregulation of PEDF in their RPE. ¹² Therefore, it is possible that the reduction of neprilysin mRNA expression induces angiogenesis. VEGF, which increases in AD patients, binds to Aβ with high affinity, ⁶⁰ and Aβ stimulates the angiogenic response through FGF-2. ⁶¹ In addition, Aβ-treated ARPE19 cells display an upregulation of VEGF and a downregulation of PEDF. ¹² Taken together, these reports suggest the possibility that Aβ induces angiogenesis. However, in our study, it was unclear whether Aβ or neprilysin directly affected angiogenesis because the stress-inducer treatment time was short. 

In the present study, we demonstrated that ER stress could cause the promotion of Aβ accumulation-inducible and angiogenic factors at the mRNA level in ARPE19 cells. Moreover, we showed that TG induced those factors more effectively than TM. Therefore, we propose that TG treatment produced an elevation of the intracellular Ca concentration, which was one of the responsible factors for the onset or acceleration of Aβ accumulation and angiogenesis in the retinal pigment epithelial cultured cells. Because Aβ exists in the RPE of patients with AMD and caused a disruption of cellular Ca homeostasis, ^{62 63} we speculate that intracellular Ca homeostasis disturbance in RPE may be involved in one of the pathogenic mechanism of AMD (Fig. 6) . Detailed analysis of the intracellular responses of ARPE19 cells to TG might be useful for the mechanism of AMD. 

 Submitted for publication August 15, 2007; revised December 26, 2007; accepted April 11, 2008.

 Disclosure: Y. Koyama, None; S. Matsuzaki, None; F. Gomi, None; K. Yamada, None; T. Katayama, None; K. Sato, None; T. Kumada, None; A. Fukuda, None; S. Matsuda, None; Y. Tano, None; M. Tohyama, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primer Sequences Used in This Study

Gene	Oligo Name	Sequence	Accession No.
APP	Forward	ctg gcc ctg gag aac tac atc a	NM_000484
	Reverse	gcg cgg aca tac ttc ttt agc a
BACE	Forward	tca ccc aag gtc acc aaa caa c	NM_012104
	Reverse	tga agt cct cac cct ttc cca t
caspase-4	Forward	ctg aag gac aaa ccc aag gtc a	U25804
	Reverse	cac ttc caa gga tgc tgg aga g
ECE	Forward	aac tcc aac agc aac gtg atc c	NM_001397
	Reverse	cgg tca gca cct tct cgt ttt
GAPDH	Forward	cac tga atc tcc cct cct cac a	NM_002046
	Reverse	tga tgg tac atg aca agg tgc g
neprilysin	Forward	cat cgg cat ggt cat agg aca	NM_007287
	Reverse	tgt tga gtc cac cag tca acg a
PEDF	Forward	gcc agg tcc aca aag gaa att	AF_400442
	Reverse	aac ttt gtt acc cac tgc ccc
TACE	Forward	ttc act gga cac gtg gtt ggt	NM_002046
	Reverse	ggc ccc atc tgt gtt gat tct
TNF-α	Forward	aac aac cct cag acg cca cat	NM_000594
	Reverse	cag tgc tca tgg tgt cct ttc c
VEGF	Forward	aag aag cag ccc atg aca gct	NM_001025366
	Reverse	tag gtc ctt tta ggc tgc acc c

Figure 1.

 

(A) Transcriptional expression of APP isoforms in cultured ARPE19 cells induced by TG, TM, and STS by RT-PCR analysis and densitometric analyses. Data are expressed as the mean ± SE (n = 3). The results show that TG and TM treatment induced a slight increase in APP751 and APP770 but not APP695 compared with the control. **P < 0.05 and *P < 0.01 for comparison with the control, by Student’s t-test. (B) Expression of APP mRNA induced by TG, TM, and STS using real-time RT-PCR analysis. Data are expressed as mean ± SE (n = 3). Expression of APP did not change in all ARPE19 cell samples compared with the control. *P < 0.01 for comparison with the control, by Student’s t-test.

Figure 2.

 

Expression of Aβ-metabolic process-related genes such as neprilysin (A), ECE (B), and BACE (C) mRNA induced by TG, TM, and STS using real-time RT-PCR analysis. Data are expressed as mean ± SE (n = 3). Expression of ECE (B) and BACE (C) did not change in all ARPE19 cells sample compared with the control. Expression of neprilysin (A) mRNA decreased in only TG-treated ARPE19 cells compared with the control. *P < 0.01 for comparison with the control, by Student’s t-test.

Figure 3.

 

Expression of angiogenesis-related genes such as VEGF (A), PEDF (B), TNF-α (C), and TACE (D) induced by TG, TM, and STS using real-time RT-PCR analysis. Data are expressed as mean ± SE (n = 3). Graphs show the upregulation of VEGF (A), TNF-α (C), and mRNA in all ARPE19 cell samples compared with the control. Expression of PEDF (B) decreased in TG- and STS-induced ARPE19 cell mRNA compared with the control. Expression of TACE (D) increased in TG- and TM-induced ARPE19 cell mRNA compared with the control. **P < 0.05 and *P < 0.01 for comparison with the control, by Student’s t-test (E) Expression of VEGF protein induced by TG, TM, and STS using Western blot. Results show the upregulation of VEGF as it was affected by TG and TM compared with the control. These data were confirmed by three independent Western blot analyses.

Figure 4.

 

(A) Expression of caspase-4 induced by TG, TM, and STS using real-time RT-PCR analysis. The graph shows the upregulation of caspase-4 mRNA as it was affected by TG compared with the control. (B) Expression of the active-form of caspase-4 (cleaved-caspase-4) induced by TG, TM, and STS using Western blot and densitometric analyses. Results show the activation of caspase-4 as it was affected by TG and TM compared with the control. Data are expressed as mean ± SE (n = 3). *P < 0.01 for comparison with the control, by Student’s t-test.

Figure 5.

 

Effects of TG and TM on Ca²⁺ elevation in retinal pigment epithelial cell soma. (A) A representative experiment showing that TG (1 μM) evoked increases in cytosolic Ca²⁺. These images are pseudocolor images of Ca²⁺ indicator dye fluorescence (a, 0 min; b, 20 seconds after TG application). Scale bar, 50 mm. (B) Representative traces showing the average changes in Ca²⁺ indicator dye fluorescence of the retinal pigment epithelial cells. Spontaneous Ca²⁺ elevations were also observed in retinal pigment epithelial cells before the application of drugs. Note that the application of TG rapidly and markedly increased Ca²⁺ indicator dye fluorescence. (C) Average amplitudes of first peaks after drug application. Note that TG significantly increased the peak amplitude of Ca²⁺ elevation but that TM did not. Statistical differences were established using the Mann-Whitney U test at **P < 0.01 (n = 34–58). Error bars indicate SEMs.

Figure 6.

 

Display of the hypothesis that the disturbances in Ca homeostasis involve the pathogenic mechanism of AMD.

Supplementary Materials

Supplementary Figure S1 - (PDF)