Alkylphosphocholines Inhibit Proliferation of Human Retinal Pigment Epithelial Cells

Abstract

purpose. To investigate the effect and mechanism of action of alkylphosphocholines (APCs) on proliferation of human retinal pigment epithelium (RPE) cells and RPE-mediated collagen matrix contraction in vitro.

methods. Cultured RPE cells of five human donors were treated with four APCs in the presence of fetal calf serum. Proliferation was assessed by the tetrazolium dye-reduction (MTT) assay and by counting the number of cells dividing in culture. The effect of APCs on RPE-mediated matrix contraction was determined in three-dimensional collagen gels. Cell viability was tested by the trypan blue exclusion assay. As a possible mechanism of APC action, protein kinase C (PKC) activity was quantified by scintillation counting of ³²P-labeled phosphate transferred to a PKC-specific substrate.

results. All APCs inhibited RPE proliferation and RPE–mediated collagen matrix contraction in a dose-dependent manner in vitro. The antiproliferative and anticontractile effect of APCs increased with elongation of the fatty acid chain beyond C20. IC₅₀s of all APCs varied between 8.5 μM (erucyl-phosphocholine, C22:1-PC), 9.0 μM (Z)-12-heneicosenyl-phosphocholine, C21:1-PC), 11.0 μM (Z)-10-eicosenyl-phosphocholine, C20:1-PC), and 26.5 μM (oleyl-phosphocholine, C18:1-PC). Trypan blue staining revealed a toxicity below 5% for all APCs within the concentration interval tested. PKC activity was significantly reduced by all four APCs, with C22:1-PC being the most effective.

conclusions. APCs inhibit proliferation of RPE cells and RPE-mediated matrix contraction in vitro at nontoxic concentrations. This effect seems to be exerted through inhibition of PKC activity. Therefore, APCs are promising candidates for treatment of RPE-mediated proliferative processes such as proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the most common cause of failure in retinal detachment surgery. ¹ This pathologic condition is characterized by the proliferation of a variety of cells in the vitreous cavity, followed by the production and subsequent contraction of the extracellular matrix, with formation of scarlike membranes on the retinal surfaces. ^{2 3} The epiretinal and subretinal membranes are mainly composed of retinal pigment epithelial cells plus glial and fibroblast-like cells. ⁴ Research data suggest that retinal pigment epithelial (RPE) cells are fundamental to PVR membrane contraction and act as a contractile force resulting in complex retinal detachment. ^{5 6}  

Pharmacologic prevention of PVR membrane formation has been based chiefly on antiproliferative and anti-inflammatory agents. ⁷ However, lack of efficacy and retinal toxicity have thus far prevented their routine clinical use. ⁸  

Alkylphosphocholines (APCs) are a new class of pharmacologically active agents with known antiproliferative, antitumoral, and antiparasitic properties. The chemical structure of APCs is characterized by an alkyl chain of variable length connected to phosphocholine by an ester bond. First-generation APCs have a saturated alkyl chain and were studied intensively in the 1980s. Their selective activity against experimental mammary carcinoma with very low toxicity to normal tissue made them an ideal candidate for topical treatment of cutaneous breast cancer metastasis. ⁹ One of the APC compounds, hexadecylphosphocholine, has been approved in several European countries for this indication and is marketed under the trade name Miltex (Zentaris, Frankfurt, Germany). ¹⁰ Because of considerable side effects when administered systemically, the structure of APCs has been modified by introduction of an unsaturated double bond at the center of the alkyl chain. Therefore, we used second-generation APCs in this study. 

The mode of action of APCs is mediated through the cell membrane, but has not yet been fully elucidated. Interference with signal transduction ¹¹ has been discussed as a possible mechanism of action, as well as the induction of apoptosis. ¹² Hexadecylphosphocholine has been found to inhibit protein kinase C (PKC) selectively in several cancer cell lines in vitro. ^{13 14} Because the activation of PKC seems to be an important feature in RPE-mediated collagen gel contraction, ¹⁴ selective inhibition of this pathway by APCs could offer a new perspective on PVR prevention. 

In this study, we investigated the antiproliferative and anticontractile effects of APCs on cultured RPE cells. The purpose of the study was to demonstrate for the first time that APCs inhibit RPE proliferation and RPE-mediated collagen matrix contraction at nontoxic concentrations in a well-established in vitro model of PVR. Furthermore, we tested to determine whether the PKC pathway is involved in the mechanism of action of APCs on human RPE cells. 

Materials and Methods

Alkylphosphocholines

The APCs oleyl-phosphocholine (C18:1-PC), (Z)-10-eicosenyl-phosphocholine (C20:1-PC), (Z)-12-heneicosenyl-phosphocholine (C21:1-PC), and erucyl-phosphocholine (C22:1-PC) were synthesized and kindly provided by Hansjoerg Eibl, PhD (Göttingen, Germany). All reagents were of analytical grade, as determined by high-performance liquid chromatography. The chemical structure is depicted in Figure 1 . 

All substances were dissolved in ethanol and stored at 4°C. Independent dilution series in ethanol were used to obtain final concentrations of APCs in equal volumes of ethanol. 

Human RPE Cell Culture

RPE cells were isolated from five human donors, age 16 to 74 years, within 4 to 16 hours after death, as described previously, ¹⁵ and were obtained from the Eye Bank of the Ludwig-Maximilians University (Munich, Germany). Methods for securing human tissue were humane, included proper consent and approval, complied with the Declaration of Helsinki, and were approved by the local ethics committee. 

In brief, whole eyes were rinsed with 0.9% NaCl solution, immersed in 5% polyvinyl pyrrolidone iodine, and rinsed again with the sodium-chloride solution. After removal of the anterior segment, the posterior pole of each donor eye was examined with a binocular stereomicroscope to exclude any gross retinal disease. Then, the neuroretina was carefully peeled away from the RPE layer with a fine forceps. The eyecup was rinsed with Ca²⁺- and Mg²⁺-free Hank’s balanced salt solution, and treated with 0.25% trypsin (Gibco, Karlsruhe, Germany) for 1 hour at 37°C. After aspiration of trypsin, the eyecup was filled with Dulbecco’s modified Eagle’s medium (DMEM; Biochrom, Berlin, Germany) supplemented with 20% fetal calf serum (FCS; Biochrom). The medium was then gently aspirated with a pipette to detach RPE into the medium without damaging the Bruch’s membrane. 

The cell suspension was transferred to a 50-mL flask (Falcon, Wiesbaden, Germany), containing 20 mL of DMEM supplemented with 20% FCS and maintained at 37°C in 5% carbon dioxide. All cell culture dishes used were uncoated. Epithelial origin was confirmed by immunohistochemical staining for cytokeratin with a pancytokeratin antibody (Sigma-Aldrich, Deisenhofen, Germany), as described previously (data not shown). ¹⁶ The cells were tested and found to be free of contaminating macrophages (anti-CD11; Sigma-Aldrich) and endothelial cells (anti-von Willebrand factor; Sigma-Aldrich; data not shown). 

Several primary cell cultures were grown to confluence. Subcultures between passages 3 and 6 (p3–6) were maintained with DMEM containing 10% FCS at 37°C and 5% carbon dioxide. In the present study, RPE cells of five human donors were used when cells were actively proliferating. 

Cell Proliferation Assay

The tetrazolium dye-reduction assay (MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) was used to determine cell survival and proliferation rate. RPE cells (p3–6) were seeded in 96-well plates (150 μL/well at a density of 5 × 10³ cells/well in DMEM and 10% FCS) and exposed to seven concentrations of APCs chosen to span the 50% inhibitory concentration (IC₅₀) as determined by preliminary assays. The IC₅₀ is defined as the concentration of a drug that produces a 50% reduction in cell number. The calculation of the IC₅₀ from the curves was performed as follows: Two concentrations closest to the IC₅₀ were identified and selected for a linear interpolation, which connected the mean response values of the upper and lower concentrations by a straight line. The value at the center of this interval represented the IC₅₀. The corresponding 95% confidence interval (CI) was calculated. 

The MTT test was performed as described by Mosmann ¹⁷ with some modifications. In brief, after incubation with APCs for 24 hours, medium was removed, cells were washed with PBS, and 200 μL/well MTT solution (1.5 mL MTT stock, 2 mg/mL in PBS, plus 28.5 mL DMEM) was added. RPE cells were incubated at 37°C for 1 hour. The formazan crystals that formed were dissolved by the addition of dimethyl sulfoxide (DMSO; 200 μL/well). The final concentration of DMSO in the cell culture medium was found to have no antiproliferative effect on RPE cells. Absorption was measured by a scanning multiwell spectrophotometer at 550 nm (Molecular Probes, Garching, Germany). Results from the wells were expressed as the mean percentage of control proliferation (control optical density [OD] 0.8 at 550 nm was assigned as 100% proliferation). Experiments were performed in triplicate and repeated five times. RPE cells of the same passage incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control. 

Cell Counting of Proliferating RPE Cells after Treatment with APCs

Human RPE cells (p3–6) were seeded in 24-well plates (Nunc, Wiesbaden Germany) at a density of 2 × 10⁴ cells per well in 1 mL DMEM and 10% FCS. After 24 hours, the medium was changed, and APCs were added once in DMEM and 10% FCS, as described earlier. Determination of live and dead cells by a standard cell counter was performed 24 hours later after RPE cells were trypsinized (Casy1, Model DT; Schärfe Systems GmbH, Reutlingen, Germany). At least 5000 cells were counted. Experiments were performed in triplicate and repeated five times. RPE cells of the same passage incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control. 

Trypan Blue Staining of Proliferating RPE Cells after Treatment with APCs

RPE cells (p3–6) were seeded in 24-well plates (Nunc) at a density of 2 × 10⁴ cells per well in 1 mL DMEM and 10% FCS. The medium was changed after 24 hours, and APCs were added in concentrations spanning the IC₅₀ interval between 3.0 and 42 μM (to 5‰ vol/vol ethanol in DMEM and 10% FCS). After 24 hours, RPE cells were trypsinized and stained with 2% trypan blue (1:1 vol/vol) for 5 minutes. Viable (unstained) and dead (stained) cells were counted from each well by hemocytometer (Neubauer chamber). Experiments were performed in triplicate and repeated five times. RPE cells of the same passage incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control. At least 400 cells were counted in each well. 

Collagen Matrix Contraction Assay

For analysis of cell-populated three-dimensional collagen matrix contraction, the method of Mazure and Grierson ⁶ was modified. Rat tail type I collagen (Sigma-Aldrich) was dissolved in 0.1% (vol/vol) acetic acid in sterile distilled water and stored at 4°C overnight. The 24-well plates were preincubated with 2% bovine serum albumin in PBS overnight to block unspecific binding. ¹⁸  

RPE cells (p3–6) were counted and resuspended in modified Eagle’s medium (MEM; Biochrom) at a volume of 1 mL containing 1 × 10⁶ cells, sufficient for one 24-well plate. The cell suspension was mixed with 5.0 mL of 3 mg/mL collagen and with 3.0 mL of concentrated serum-free MEM containing glutamine, antibiotics, and 391 μL 1 M NaOH. The collagen–cell mixture was then transferred in 350-μL aliquots to a 24-well plate to cover the bottom of the wells. The solution polymerized within 1 hour when incubated at 37°C. Trapping the cells at a density of 4 × 10⁴ RPEs per matrix, the three-dimensional collagen gels were detached from the bottom of the wells after 1.5 hours. The matrices were floated in 1 mL DMEM and 10% FCS containing one of the four APCs at its IC₅₀ (to 5‰ vol/vol ethanol). The test was performed in triplicate and repeated five times. RPE cells of the same passage incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control. 

After 24 hours, the media were removed and the gels were washed and incubated with DMEM and 10% FCS. The 24-well plates were then incubated at 37°C for another 6 days. The media were changed every other day. Collagen gels without addition of RPE cells were used for calculation of baseline contraction. The surface area of each matrix was observed, recorded, and measured digitally (LAS-1000 Imager; RayTest, Pforzheim, Germany) every third day. The percentage of gel contraction was calculated as [(gel size at day 1 − gel size at day 6)/gel size at day 1] × 100. 

Measurement of PKC Activity

For determination of PKC activity, a radioactive assay was applied to investigate the mechanism of action of APCs on RPE cells in vitro (SignaTECT Protein Kinase C Assay System; Promega, Madison, WI). ¹⁹ This assay is based on the measurement of ³²P-labeled phosphate transfer to a PKC-specific peptide that can be captured on phosphocellulose filters. It is known to be PKC specific and reliable for measurement of enzyme activity in crude tissue extracts. ²⁰  

RPE cells (p3–6) were seeded in 35-mm diameter Petri dishes at a density of 1 × 10⁵ cells per dish in DMEM and 10% FCS, exposed to one of the four APCs for 24 hours, and processed as indicated by the manufacturer’s protocol. In brief, after incubation with APCs at a concentration interval spanning the IC₅₀, as determined in preliminary assays, the media were removed and the cells were washed with PBS, resuspended, and homogenized (40 strokes; Dounce homogenizer; Bellco Glass Co., Vineland, NJ) in cold extraction buffer (25 mM Tris-HCl [pH 7.4]; 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM β-mercaptoethanol, 1 μg/mL leupeptin, and 1 μg/mL aprotinin). Cell lysates were passed over a 1 mL column of diethylaminoethyl (DEAE) cellulose (DE52; Whatman, Kent, UK) prepared previously. To elute the PKC-containing fraction, 5 mL of extraction buffer containing 200 mM NaCl was used. The amount of protein per enzyme sample was determined as described by Bradford ²¹ (Bio-Rad, Mannheim, Germany). 

After incubation of the enzyme sample with the PKC biotinylated peptide substrate and [³²P] adenosine triphosphate (ATP) in the appropriate reaction volume at 30°C for 5 minutes, the reaction was terminated, and 10 μL of the reaction volume was spotted on a streptavidin-labeled membrane (SAM² membrane) which was supplied with the PKC assay system. Membranes were washed and dried according to the manufacturer’s protocol before analysis by scintillation counting was performed. The test was performed in triplicate and repeated five times. RPE cells of the same passage incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control. To further ensure PKC specificity of the assay, we used a commercial PKC inhibitor on control PKC activity (Myristoylated PKC Peptide Inhibitor; Promega) which reduced control PKC activity to background level (control PKC activity: 1.1 × 10⁶ counts; background: 0.2 × 10⁶ counts; data not shown). 

Statistical Evaluation

Statistical analysis was performed on computer (SPSS, ver. 11.0; SPSS Science Inc., Chicago, IL). All results are expressed as the mean ± SEM or 95% CI, as indicated. For determination of the significance of differences, an ANOVA was performed. Differences at P < 0.01 were considered statistically significant. 

Results

Inhibition of RPE Proliferation by APCs In Vitro

APCs inhibited proliferation of human retinal pigment epithelium in a dose-dependent manner in vitro (Fig. 2) . The actual number of cells correlated well with the results of the MTT test. A single application of each APC and continuous exposure of RPE cells over 24 hours prevented an increase in the number of cells (ANOVA analysis; P < 0.001). The observed effect was modulated by structural variations of APCs. An elongation of the fatty acid chain beyond C20 led to an increase in antiproliferative efficacy, as indicated by smaller IC₅₀s (Table 1) . For further comparison of the four APCs, the concentration that inhibited the control cell growth by 50% (IC₅₀) was determined from each of the dose–response curves, respectively. 

Oleyl-phosphocholine (C18:1-PC; Fig. 1A ) showed an inhibition of RPE cell proliferation at concentrations between 10 and 42 μM (Fig. 2) . Cells exposed to C18:1-PC at concentrations below 10 μM were not compromised in their growth behavior compared with the control. The IC₅₀ was 26.5 μM ± 1.3 CI (Table 1) and differed significantly from the IC₅₀s of all other APCs applied (post hoc pair-wise comparison, P < 0.01). 

Eicosenyl-phosphocholine (C20:1-PC; Fig. 1B )–treated RPE cells decreased in number at concentrations higher than 4.2 μM (Fig. 2) . C20:1-PC concentrations lower than 3 μM yielded cell counts similar to the control. The IC₅₀ was 11.0 μM ± 0.7 CI (Table 1) . Heneicosenyl-phosphocholine (C21:1-PC; Fig. 1c ) displayed a dose–response curve and corresponding IC₅₀ close to that of C20:1-PC. For C21:1-PC, the IC₅₀ was 9.0 μM ± 0.9 CI (Table 1) . IC₅₀ s of both substances did not differ significantly (P > 0.01). 

Erucyl-phosphocholine (C22:1-PC; Fig. 1d ) inhibited RPE cell proliferation at concentrations higher than 3 μM (Fig. 2) . For C22:1-PC, the IC₅₀ was 8.5 μM ± 2.3 CI (Table 1) and did not differ significantly from the IC₅₀ of C20:1-PC nor from that of C21:1-PC. 

The calculated IC₅₀s based on the dose–response curves for each APC indicated differences in efficacy as follows. The IC₅₀ of C18:1-PC exceeded 20 μM, indicating the lowest efficacy of all APCs applied. The IC₅₀s of C20:1-PC, C21:1-PC, and C22:1-PC were quite close together at approximately 10 μM. On comparison of the concentration-dependent antiproliferative effect of the four APCs, substances with a fatty acid chain of C20 or longer displayed a higher antiproliferative efficacy compared with C18:1-PC. 

Cell Viability Study

Proliferating RPE cells were treated with the four APCs at concentrations between 3 and 42 μM. This interval was chosen to span the IC₅₀ of each APC and to determine cell viability at effective antiproliferative and anticontractile concentrations in vitro. Cell morphologic changes in phase-contrast microscopy and toxicity, as determined by the trypan blue exclusion test, did not differ from the control cells which corresponded to a maximum toxicity (blue staining) of 5% of APC-treated RPE cells (Table 1) . 

Inhibition of Contraction of RPE-Populated Collagen Matrices by APCs

Human RPE cells were used to populate collagen gel matrices. All four APCs caused a significant concentration-dependent inhibition of RPE cell–mediated collagen gel contraction in the presence of 10% fetal calf serum (Fig. 3) . The baseline RPE-mediated collagen gel contraction was 46% of the control. Incubation with APCs at their IC₅₀s decreased collagen gel contraction markedly. After treatment, C18:1-PC and C20:1-PC collagen gel contraction was reduced to 12% of control level and to only 11% after incubation with C21:1-PC and C22:1-PC. Thus, baseline RPE-mediated collagen contraction was reduced by 74% (C18:1-PC and C20:1-PC, respectively) and by 76% after treatment with (C21:1-PC and C22:1-PC, respectively). 

Inhibition of PKC Activity in APC-Treated RPE Cells

Our data demonstrate that all four APCs, at their IC₅₀s, inhibited PKC activity in proliferating RPE cells effectively as determined by a radioactive assay (Fig. 4) . Control PKC activity was set to 100%. 

Oleyl-phosphocholine (C18:1-PC) reduced PKC activity to 59% at its IC₅₀. The concentration interval tested varied between 7.5 μM (84% PKC activity) and 42 μM (19% PKC activity). Spanning the IC₅₀s of C20:1-PC, C21:1-PC, and C22:1-PC, we chose concentrations between 3 and 18 μM to test APC-induced inhibition of PKC activity. At their IC₅₀s, PKC inhibition varied between 52% (C20:1-PC), 46% (C21:1-PC), and 43% (C22:1-PC). Maximum inhibition of PKC activity was achieved by 18 μM C22:1-PC to a minimum of 7% compared with 14% (C21:1-PC) and 16% (C22:1-PC) at equal concentrations. The IC₅₀s calculated for PKC inhibition are 22.5 μM for C18:1-PC, 10.6 μM for C20:1-PC, and 9.8 μM for both C21:1-PC and C22:1-PC. Thus, the IC₅₀s calculated for proliferation inhibition by APCs correlate well with the ones for PKC inhibition. 

Discussion

Prevention of PVR membrane formation has been the goal of numerous studies that sought to preserve vision in eyes with complicated retinal disorders. However, considerable side effects have thus far prevented the application of conventional anticancer drugs. ^{22 23 24} To date, none of several proposed PVR treatment regimens is in routine clinical use. ⁷  

APCs, a new class of pharmacologically active agents, have been reported to show only marginal side effects at antineoplastic concentrations for the treatment of cutaneous breast cancer metastasis. ^{25 26 27} The antiproliferative effect at low toxicity has also prompted their application for treatment of visceral leishmaniasis. ²⁶ From a chemical point of view, APCs are simple structures containing long chain fatty alcohols esterified with phosphocholine. These molecules have the physical properties of lysolecithins and rapidly incorporate in biological membranes starting from a minimum alkyl chain length of C16. An important structural parameter of second-generation APCs is the introduction of a cis-double bond in the middle of the alkyl chain. It simultaneously reduces toxicity and improves antineoplastic and antiprotozoal activity. ²⁸ These results obtained in a different field of research prompted us to use APCs with one cis-double bond for the treatment of RPE cells in the present in vitro study. 

Our results demonstrate that all tested APCs significantly inhibited RPE proliferation in a dose-dependent manner. The viability in all RPE cell cultures treated with APCs at effective antiproliferative and anticontractile dosages was equivalent to the control. Because PVR membrane disease depends to a great extent on RPE cell proliferation and contractile properties, the antiproliferative and anticontractile effect of APCs strongly supports their possible role as agents for PVR prevention. However, APC-mediated inhibition of collagen contraction could be partly related to simultaneous inhibition of RPE proliferation in collagen gels. 

Regarding the mechanism of action, it is known that APCs affect protein kinase function as part of the second-messenger system and probably also as part of the cell-cycle control system. It has been shown that APCs inhibit PKC function in several cancer cell lines. ^{13 29 30 31} PKC is a membrane-bound G-protein involved in the intracellular cascade of second-messenger systems regulating major cellular functions like proliferation, differentiation and cell division. Functions known to involve the PKC pathway in RPE cells are the cell-mediated collagen gel contraction ¹⁴ and cellular migration. ³² Our data demonstrate that APCs inhibit control PKC activity to a great extent in the presence of serum. Other PKC inhibitors, such as calphostin C and staurosporine, have been far less effective in PKC inhibition in the presence of serum than in serum-free conditions. ³³  

The classic PKC isoforms α, β1, and β2 are all expressed by human RPE cells and need cis-unsaturated fatty acids, phosphatidylserine, and diacylglycerol for activation. ³⁴ This implies a possible role for APCs through interaction with phosphatidylserine and/or cis-unsaturated fatty acids during PKC activation. We thus propose the inhibition of PKC as a possible mechanism of action by which APCs exert antiproliferative and anticontractile effects on human RPE cells. At the same time, we are aware that other signaling pathways could be involved in the mechanism of action of APCs in human RPE cells. 

Based on our observations, the intravitreal application of APCs in vivo seems feasible. In extending our in vitro results to clinical trials, safety considerations regarding retinal side effects and activity in the presence of serum become an issue. Because all experiments were performed in the presence of serum, APC activity should not be compromised in the vivo situation where the breakdown of the blood–retinal barrier, one of the main features of PVR, leads to a dispersion of serum components in the vitreous cavity. In the in vitro situation, the presence of serum is known as a strong stimulator of RPE cell proliferation, ³⁰ and even in this situation, APCs have been effective in exerting their inhibitory effect on RPE cell proliferation and RPE-mediated collagen matrix contraction. 

Regarding comparison of the concentration-dependent antiproliferative effect of the four APCs, the substance with the highest efficacy (C22:1-PC) seems to be the best choice for possible in vivo application. C22:1-PC has also been well tolerated when given intravenously to the rat for potential treatment of malignant brain tumors in dosages of 20 mg/kg over 4 weeks. ³¹ The therapeutic range of all APCs applied to RPE in vitro is within a log range and thus rather small. Further experiments are needed to determine effectiveness and tolerance in vivo. 

In summary, our results demonstrate that APCs had antiproliferative and anticontractile effects on human RPE cells at concentrations that were nontoxic to the cells. An elongation of the carbon chain led to an increase in these effects. The mechanism of action of APCs in human RPE cells seems to be mediated by inhibition of the PKC pathway. APCs represent a new class of pharmacologically active agents and are a promising candidate for prevention of PVR. 

 Supported by German Research Foundation Grant WE 2577/2-1 and the Vera und Volker Doppelfeld Foundation.

 Submitted for publication November 16, 2002; revised March 2, 2003; accepted March 4, 2003.

 Disclosure: K.H. Eibl, None; B. Banas, None; C.-L. Schoenfeld, None; C.A. May, None; A.S. Neubauer, None; S. Priglinger, None; A. Kampik, None; U. Welge-Lussen, 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.

 

Figure 1.

 

Chemical structure of the four APCs used in this study. (A) Oleyl-phosphocholine (C18:1-PC); (B) (Z)-10-eicosenyl-phosphocholine (C20:1-PC); (C) (Z)-12-heneicosenyl-phosphocholine (C21:1-PC); (D) erucyl-phosphocholine (C22:1-PC).

Figure 2.

 

Inhibition of retinal pigment epithelium (RPE) cell proliferation by APCs measured by a colorimetric test (MTT; A) and by cell counting (B). The MTT test measures the activity of the mitochondrial enzyme succinate tetrazolium reductase, which is directly related to the number of viable cells. Human RPE cells (p3–6) were cultured in 96-well plates at a density of 5 × 10³ cells per well for the MTT test (A) and in 24-well plates at a density of 2 × 10⁴ cells per well to obtain cell counts (B) in DMEM and 10% FCS. After 24 hours, medium was changed and APCs were added. The concentrations varied between 3 and 42 μM to span the IC₅₀, as determined by preliminary assays. This is the concentration of a drug that produces a 50% reduction in cell number. The IC₅₀ was calculated based on results of an MTT test (A). The tests were performed in triplicate and repeated five times. RPE cells of the same passage incubated with equal volumes of ethanol without addition of APCs served as the control. Results were expressed as the mean percentage of control proliferation (control OD 0.8 at 550 nm was assigned as 100% proliferation for MTT). Data are the mean of results in five experiments, each performed in triplicate. Error bars, SEM.

Table 1.

 

IC₅₀ and Maximum Concentration and Toxicity of APCs

APC	IC50 Concentration (μM ± CI)	Maximum Concentration (μM)	Maximum Toxicity (% ± SEM)
C18:1-PC	26.5 ± 1.3	42.0	5 ± 3
C20:1-PC	11.0 ± 0.7	18.0	5 ± 1
C21:1-PC	9.0 ± 0.9	18.0	5 ± 3
C22:1-PC	8.5 ± 2.3	13.0	2 ± 1

Figure 3.

 

RPE cell-mediated contraction of populated collagen matrices after treatment with the APCs C18:1-PC, C20:1-PC, C21:1-PC, and C22:1-PC as percentage of gel contraction compared to collagen only. Human RPE cells (p3–6) were seeded at a density of 1 × 10⁶ cells per 24-well plate in concentrated serum-free MEM and mixed with 3 mg/mL collagen. After polymerization of these RPE-populated collagen matrices, DMEM and 10% FCS were added with one APC per well at its IC₅₀ to float the gel matrices. RPE-mediated collagen gel contraction was observed daily, recorded, and measured digitally on day 6. RPE cells of the same passage incubated with equal volumes of ethanol without addition of APCs served as the control. Data are the mean of results in five experiments, each performed in triplicate. Error bars, SEM.

Figure 4.

 

PKC activity was measured with a radioactive assay. RPE cells (p3–6) were seeded at a density of 1 x×10⁵ cells in a 35-mm Petri dish and treated with one APC per dish at a concentration interval spanning the IC₅₀. Cell lysates were passed over a cellulose column and the PKC-containing fraction eluted. Control PKC activity was set to 100% (1.1 × 10⁶ counts; background: 0.2 × 10⁶ counts). Data are the mean of three experiments, each performed two times. Error bars, SEM.

The authors thank Ken R. Kenyon, MD, for careful review of the manuscript and helpful discussion and Katja Obholzer for expert technical assistance.