Alkylphosphocholines: A New Therapeutic Option in Glaucoma Filtration Surgery

Abstract

purpose. To investigate the effect of alkylphosphocholines (APCs) on human Tenon fibroblast (HTF) proliferation, migration, and cell-mediated collagen gel contraction.

methods. HTFs were isolated from tissue samples of three patients obtained during surgery and cultured in DMEM and 10% fetal calf serum (FCS). HTFs (passage 3–6) were treated with one APC in different concentrations spanning the 50% inhibitory concentration (IC₅₀), as determined previously. Inhibition of cell proliferation was assessed by the tetrazolium dye reduction assay. Migration was determined in chemoattractant chambers with fibronectin-coated polycarbonated membranes. For inhibition of contraction, three-dimensional collagen gels were seeded with HTFs, and the gel size was measured. Cell viability was determined by the trypan blue exclusion assay. For analysis of the mechanism of action, protein kinase C (PKC) activity was measured.

results. The IC₅₀ varied between 7.0 and 10.5 μM for all APCs tested. At this concentration, all four APCs inhibited HTF migration and cell-mediated collagen gel contraction in the presence of serum. The inhibitory effects on HTF proliferation, migration, and contraction were observed at nontoxic concentrations. PKC activity was reduced to 50% of control level at the IC₅₀ of all APCs applied.

conclusions. APCs are effective inhibitors of HTF proliferation, migration, and cell-mediated contraction of collagen gels at nontoxic concentrations. Their mechanism of action seems to involve the inhibition of the PKC pathway.

Glaucoma is a major cause of blindness worldwide. Despite effective antiglaucoma medication, a large number of patients with primary open-angle or closed-angle glaucoma have to undergo trabeculectomy to preserve vision during the course of the disease. ^{1 2 3}  

The main cause of failure in glaucoma filtration surgery is the scarring of the filtering bleb site. ⁴ Fibroblasts from the subconjunctival space play a crucial role in this process through proliferation, migration, production, and subsequent contraction of the extracellular matrix. ⁵ Current concepts of therapy are intended to limit the scarring process and include the perioperative administration of antimetabolites such as 5-fluorouracil (5-FU) and mitomycin C (MMC). ^{6 7 8} These agents allow for better control of the scarring complications but are accompanied by severe side effects, such as keratitis, bleb leakage, chronic hypotony with maculopathy, and endophthalmitis. ^{9 10 11 12 13} Despite the use of these antiproliferative agents, surgery can still fail in some patients, probably because of the tendency of the growth-arrested fibroblasts to migrate and interact with surrounding untreated fibroblasts. ^{5 14 15 16 17 18} Recently, anti-TGF-β2 antibodies have been launched as a new antiscarring treatment option in glaucoma filtration surgery. ^{4 19 20 21} Clinical studies are under way, but neutralizing anti-TGF-β2 antibodies are not yet available for routine clinical use. 

Alkylphosphocholines (APCs) represent a new class of substances with antiproliferative properties. Hexadecylphosphocholine (miltefosine; Cayman Chemical Co., Ann Arbor, MI) is known as an effective treatment of cutaneous breast cancer metastasis due to its low toxicity to the surrounding tissue compared with 5-FU. ^{22 23} In a recent study, we were able to show an inhibitory effect of APCs on proliferation and matrix contraction in cultured retinal pigment epithelial cells. ²⁴ As these cellular mechanisms are also involved in the scarring process after glaucoma surgery, APCs could be an interesting alternative for antiscarring therapy in glaucoma filtration surgery without the disadvantages of 5-FU and MMC. 

In our study, we used four APCs in different concentrations in the presence of serum to test their ability to inhibit proliferation, migration, and contraction of human Tenon fibroblasts (HTFs) at nontoxic concentrations. We also investigated their mechanism of action with the main focus on protein kinase C (PKC), because this has been proposed in other cell systems. ^{25 26 27}  

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 (Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany). All reagents were of analytical grade as determined by high-performance liquid chromatography. 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. 

HTF Isolation and Cell Culture Conditions

HFT samples were obtained from tissue explants of three white male patients (age 20, 40, and 60 years) without any topical eye treatment, who underwent routine cataract or strabismus surgery. Informed consent was obtained from the subjects after explanation of the nature and possible consequences of the procedure. The HTFs were cultured as previously described ⁷ and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (vol/vol) FCS, 2 mM l-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, 50 mg/mL gentamicin, and 0.25 mg/mL amphotericin B (all from Invitrogen-Gibco, Paisley, Scotland, UK) at 37°C with 5% (vol/vol) CO₂ in air. Cultures were used between passages 3 and 6 for all experiments. The guidelines of the Declaration of Helsinki were followed, and institutional human experimentation committee approval was granted. 

Cell Proliferation Assay

The tetrazolium dye-reduction assay (MTT; (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma-Aldrich, St. Louis, MO) was used to determine cell survival and proliferation rate. HTFs (passages 3–6) were seeded in 96-well plates (150 μL/well at a density of 5 × 10³ cells/well in DMEM and 10% fetal calf serum [FCS]) and exposed to five 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 the number of cells. 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 IC_50. The corresponding 95% confidence interval was calculated. 

The MTT test was performed as described by Mosmann ²⁸ with some modifications. In brief, after incubation with APCs for 48 hours, the cell culture medium was removed, and the cells were washed with PBS. The MTT solution (1.5 mL MTT stock [2 mg/mL in PBS] plus 28.5 mL DMEM) was added (200 μL/well). HTFs were incubated at 37°C for 1 hour. Formazan crystals that formed were dissolved by the addition of DMSO (200 μL/well). The final concentration of DMSO in the cell culture medium was found to have no antiproliferative effect on HTFs. Absorption was measured by a scanning multiwell spectrophotometer at 550 nm (Molecular Probes, Garching, Germany). Results from the wells are expressed as the mean percentage of control proliferation (control OD 0.7 at 550 nm assigned as 100% proliferation). Experiments were performed in triplicate and repeated five times. HTFs of the same passage number incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control. 

Trypan Blue Staining of Proliferating HTFs after Treatment with APCs

HTFs (passages 3–6) 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. The medium was changed after 24 hours, and APCs were added in concentrations spanning the IC₅₀ interval between 0.1 and 42.0 μM (to 5‰ [vol/vol] ethanol in DMEM and 10% FCS). After 48 hours, HTFs were trypsinized and stained with 2% trypan blue (1:1 vol/vol) for 5 minutes. Viable (unstained) and dead (stained) cells were counted in each well with a hemocytometer (Neubauer chamber). Experiments were performed in triplicate and repeated five times. HTFs of the same passage number incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control. At least 400 cells from each well were counted. 

Cell Migration Assay

HTF migration was assayed by a modification of the Boyden chamber method ²⁹ in microchemotaxis chambers (NeuroProbe, Gaithersburg, MD) with polycarbonate filters (Nucleopore, Karlsruhe, Germany) with a pore size of 8.0 μm. The filters were coated with fibronectin (25 ng/mL in PBS; Sigma-Aldrich, Deisenhofen, Germany) and placed between the chambers. First, the lower half of the chamber was filled with 190 μL DMEM after TGF-β2 (10 ng/mL; Sigma-Aldrich) was added. ¹⁹ HTFs (passages 3–6) were trypsinized and suspended at a concentration of 5 × 10⁵ cells/mL in DMEM supplemented with 0.4% FCS. The cells were mixed with APCs in four concentrations (0.1, 1.0, 10, or 30 μM) and placed in the upper half of the chamber (100 μL/chamber). The chambers were incubated at 37°C and 5% CO₂ (vol/vol) in air for 15 hours. The filters were then removed and the HTFs on the upper side of the filter were scraped off with a cotton tip. The migrated cells on the other side of the filter were fixed in methanol and stained with hematoxylin and eosin. Five randomly chosen fields were counted at a 200× magnification with a phase-contrast microscope (Leica, Wetzlar, Germany). Experiments were performed in triplicate and repeated at least three times. HTFs of the same passage number incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control (number of control cells, 218 ± 12). Results are expressed as the percentage of the number of control cells. 

Collagen Lattice Contraction Assay

For analysis of cell-populated, three-dimensional collagen matrix contraction, the method of Mazure and Grierson ³⁰ was modified. ^{31 32} 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. 

HTFs (passages 3–6) were counted and resuspended in modified Eagle’s medium (MEM; Biochrom, Berlin, Germany) 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 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. Having trapped the cells at a density of 4 × 10⁴ HTFs per matrix after 1.5 hours, the three-dimensional collagen gels were detached from the bottom of the wells. 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. HTFs of the same passage number incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control. 

After 72 hours, the medium was 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 medium was changed every other day. Collagen gels without addition of HTFs 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_{day 1} − gel size_{day 6})/(gel_{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 PKC Assay System; Promega, Madison, WI). ³³ 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. ³⁴  

HTFs (passages 3–6) were seeded in 35-mm Petri dishes at a density of 1 × 10⁵ cells/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 one APC per well at its IC₅₀ (to 5‰ [vol/vol] ethanol) determined in preliminary assays (Fig. 2) , the medium was removed, and the cells were washed with PBS, resuspended, and homogenized (40 strokes with a 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 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 ³⁵ (Bio-Rad, Mannheim, Germany). 

After incubation of the enzyme sample with the PKC biotinylated peptide substrate and [³²P]ATP in the appropriate reaction volume at 30°C for 5 minutes, the reaction was terminated, and 10 μL of the reaction volume spotted on a streptavidin-labeled membrane (SAM² Membrane) which was supplied with the PKC assay system (Promega). Membranes were washed and dried according to the manufacturer’s protocol before analysis by scintillation counting. The test was performed in triplicate and repeated five times. HTFs of the same passage number incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control. To ensure further the 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 V 11.0; SPSS Science, Inc., Chicago, IL). All results are expressed as the mean ± SEM or ± 95% confidence interval (CI), as indicated. For determination of the significance of differences, an ANOVA was performed. Differences with P < 0.01 were considered statistically significant. 

Results

Inhibition of HTF Proliferation by APCs In Vitro

APCs inhibited proliferation of HTFs in a dose-dependent manner in vitro (Fig. 1A) . A single application of each APC and continuous exposure of HTFs over 48 hours prevented an increase in the counted number of cells (ANOVA analysis; P < 0.001). This effect was observed with all APCs applied, regardless of different alkyl chain lengths. Consequently, the IC₅₀ did not differ significantly among the four APCs applied (P > 0.01; Table 1 ). The concentration inhibiting control cell growth by 50% (IC₅₀) was determined from each of the dose–response curves. 

Oleyl-phosphocholine (C18:1-PC) showed an inhibition of HTF cell proliferation at concentrations between 1 and 42 μM (Fig. 1) . Cells exposed to C18:1-PC at concentrations below 1 μM were not compromised in their growth compared with the control. The IC₅₀ was 10.5 ± 1.2 μM (Table 1) and did not differ significantly from the IC₅₀ of all other APCs applied (P > 0.01). However, the dose–response curve of C18:1-PC lay slightly above the ones for the other APCs with an alkyl chain length of C20 and more. 

Eicosenyl-phosphocholine (C20:1-PC)–treated HTFs decreased in number at concentrations above 0.1 μM (Fig. 1) . This corresponds well to the dose–response curves of heneicosenyl-phosphocholine (C21:1-PC) and erucyl-phosphocholine (C22:1-PC). For these APCs, concentrations below 0.1 μM yielded quantities of cells similar to the control. Their IC₅₀ values were as follows: 7.0 ± 1.1 μM for C20:1-PC, 8.0 ± 0.9 μM for C21:1-PC, and 8.5 ± 1.2 μM for C22:1-PC (Table 1) . 

Cell Viability Study

Proliferating HTFs were treated with the four APCs at concentrations between 0.1 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, which corresponded to a maximum toxicity (blue staining) of 5% in APC-treated RPE cells (Table 1) . 

Inhibition of HTF Migration by APCs

HTF migration was assessed using microchemotaxis chambers with fibronectin-coated polycarbonate filters placed on the TGF-β2/DMEM-filled lower half of the chamber. HTFs were placed in the upper half of the chamber with APCs added in different concentrations and incubated under standard cell culture conditions for 15 hours. 

All APCs applied inhibited HTF migration under serum conditions at concentrations above 0.1 μM. This corresponds to the concentration interval of the antiproliferative effect of these substances. At their IC₅₀ (Table 1) , however, all APCs inhibited HTF migration completely (Fig. 2) . Fifty-percent inhibition of HTFs was achieved at concentrations between 0.1 and 1 μM which is by a factor 10 to 100 lower than proliferation inhibition. 

Inhibition of Contraction of HTF-Populated Collagen Matrices by APCs

HTFs were used to populate collagen gel matrices. All four APCs caused a significant concentration-dependent inhibition of HTF cell-mediated collagen gel contraction in the presence of 10% FCS (Fig. 3) . The baseline HTF-mediated collagen gel contraction was 57% (control). Incubation with APCs at their IC₅₀ decreased collagen gel contraction markedly. After treatment with C18:1-PC, C21:1-PC, and C22:1-PC, collagen gel contraction was reduced to 7% (12% of control level). Incubation with C20:1-PC at its IC₅₀ reduced HTF-mediated collagen contraction to 12% (21% of control level). Thus, baseline HTF-mediated collagen contraction was reduced by 79% (C20:1-PC) and even more effectively by up to 88% (C18:1-PC, C21:1-PC, and C22:1-PC). 

Inhibition of PKC Activity in APC-Treated HTFs

PKC activity in proliferating HTFs was determined with a radioactive assay. Our data demonstrate that all four APCs inhibited PKC activity at their IC₅₀ effectively (Fig. 4) . Control PKC activity was set to 100%. 

Oleyl-phosphocholine (C18:1-PC) reduced PKC activity to 49% at its IC₅₀. This corresponds with the values for the other APCs tested at their IC₅₀: C20:1-PC reduced PKC activity to 52% as did C22:1-PC, and C21:1-PC reduced PKC activity to 50%. 

Thus, all APCs were equally effective in PKC inhibition. The IC₅₀ calculated for proliferation inhibition by APCs correlated well with the ones for PKC inhibition in HTFs. 

Discussion

Scarring is the reason for the failure of surgery in many visually disabling and blinding conditions. Successful filtration surgery for the treatment of glaucoma depends directly on an individual’s wound-healing response. ² Maintenance of intraocular pressure in the low teens can prevent long-term progression of glaucoma. ^{1 18} However, scarring of the filtering bleb site leads to an increase in intraocular pressure with further progression of glaucoma, resulting in surgical failure. ^{16 17} HTFs play a crucial role in this process. Studies have shown that subconjunctival scarring of the filtering bleb site is mainly mediated by HTF proliferation, migration, and contraction. ^{5 14}  

In the present study, for the first time APCs were found to have an antiproliferative effect on HTFs. In addition, other features of the scarring process, such as HTF migration and contraction of scarlike tissue, were significantly inhibited by APCs. Viability testing showed that this was not due to HTF cell toxicity in the concentration interval tested. 

APCs are known to have antiproliferative effects in other cell systems. ^{25 36 37} Based on these findings APCs are successfully used for the treatment of cutaneous breast cancer metastasis and visceral leishmaniasis in humans (hexadecylphosphocholine; miltefosine; Cayman Chemical). ^{23 38} Because inhibition of the proliferation of HTFs is the reason for the subconjunctival injection of such toxic antimetabolites as MMC and 5-FU after glaucoma filtration surgery, ^{7 8} the application of APCs in vivo could be a favorable alternative. However, ocular toxicity cannot be excluded, since there are no data available on toxicity so far. As far as general toxicity is concerned, gastrointestinal side effects have been observed after oral administration of first-generation APCs in humans. ³⁹ The introduction of a cis double bond into the middle of the alkyl chain of the second generation APCs used in this study reduces toxicity and at the same time improves antineoplastic and antiprotozoal activity. ^{39 40} In normal and C6 glioma-bearing rats, intravenous injections of erucylphosphocholine (C22:1-PC; 20 mg/kg given at intervals of 48 hours for up to 4 weeks) did not cause any toxic effects. ⁴¹  

Moreover, APCs are able to inhibit HTF migration, which is known as a crucial event in the development of bleb failure after glaucoma filtration surgery. ⁵ An in vivo glaucoma filtration model in the rabbit has shown that successful inhibition of HTF migration by the metalloproteinase inhibitors can prevent scarring of the filtering bleb site. ^{42 43} Because APCs can prevent HTF migration at an even lower concentration than they exert on the antiproliferative effect on HTFs in vitro, scarring of the filtering bleb site was prevented by the application of a smaller APC dose at a very early stage of the process. 

If we transfer these results to the in vivo situation, a modulation of the scarring process at different levels seems feasible. First of all, the number of HTFs at the filtering bleb site was lowered by AP- induced inhibition of migration and proliferation, resulting in a smaller number of fibroblasts capable of contracting. Furthermore, APCs prevented the contraction of scarlike tissue mediated by cells without locomotion. Because APCs had an anticontractile effect on HTF-populated collagen gels in vitro, it is likely that they would inhibit matrix contraction after glaucoma filtration surgery in vivo. 

In general, it is believed that major cellular functions such as migration, proliferation, and contraction are mediated at least in part by PKC. ^{44 45 46} PKC is a membrane-bound G-protein involved in the intracellular cascade of second-messenger systems that regulate these major cellular functions. However, these cellular processes are also involved in the scarring process after glaucoma filtration surgery. 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. ²⁵ To our knowledge, involvement of PKC in the scarring process of glaucoma filtration surgery mediated by HTFs has not been described. Thus, for the first time, we were able to show PKC activity in untreated cultured HTFs. Treatment of HTFs by APCs inhibits PKC activity to a great extent, in accordance with other cell systems, such as several cancer cell lines and human retinal pigment epithelium cells. ^{24 26}  

Our results have shown that PKC activity and the major cellular functions of HTFs involved in the scarring process, such as proliferation, migration, and contraction were inhibited by APCs. All these effects were achieved after a single application of APCs and in the presence of serum. Because several growth factors are present in the aqueous humor of patients with glaucoma, the performance of experiments under serum conditions is close to the in vivo situation. Previously, it has been shown that TGF-β is the most potent stimulator of HTF activity ⁴⁷ and that the aqueous humor of glaucomatous eyes contains increased levels of TGF-β2. ^{48 49} After glaucoma surgery, elevated levels of activated TGF-β2 at the filtering bleb site are therefore likely to be related to the concentration found in the aqueous humor, due to its flow and the breakdown of the blood–aqueous barrier. The concentrations of TGF-β2 in the aqueous humor of patients with primary open-angle glaucoma are comparable to that of FCS-containing medium (1.6 ng/mL). This is of special importance, because wound healing in glaucoma filtration surgery is either influenced by aqueous humor or by the breakdown of the aqueous–blood barrier during surgery. Therefore, experimental conditions without serum-containing medium would not be comparable to the in vivo situation found in glaucoma filtration surgery. 

In summary, our results demonstrate that APCs inhibit HTF proliferation, migration, and contraction at concentrations that are nontoxic to these cells. The mechanism of action of APCs on HTFs seems to involve the inhibition of the PKC pathway. Thus, APCs may be promising for prevention of bleb failure in glaucoma filtration surgery. Further experiments are needed to determine effectiveness and tolerance in vivo. 

 Supported by Deutsche Forschungsgemeinschaft (DFG) Grant WE 2577/2-1.

 Submitted for publication December 14, 2003; revised March 5, 2004; accepted March 14, 2004.

 Disclosure: K.H. Eibl, None; B. Banas, None; D. Kook, None; A.V. Ohlmann, None; S. Priglinger, None; A. Kampik, None; U.C. Welge-Luessen, 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.

 

Inhibition of HTF cell proliferation by APCs measured by a colorimetric test (MTT; A) and by cell counting (B). The tests were performed in triplicate and repeated five times. HTFs of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control. Results were expressed as the mean percentage of control proliferation. Data are the mean of result in five experiments, each performed in triplicate. Error bars, SEM.

Table 1.

 

Summary of the IC₅₀ for Each APC and Maximum APC Concentrations Applied with the Corresponding HTF Cell Toxicity

APC	IC50 Concentration (in μM ± CI)	Maximal Concentration (μM)	Maximal Toxicity (in % ± SEM)
C18:1-PC	10.5 ± 1.2	42.0	4 ± 2
C20:1-PC	7.0 ± 1.1	42.0	2 ± 1
C21:1-PC	8.0 ± 0.9	42.0	3 ± 2
C22:1-PC	8.5 ± 1.2	42.0	5 ± 2

 IC₅₀ is expressed in mean micromolar ± 95% confidence interval (CI), and cell toxicity is expressed as percent of toxicity ± SEM.

Figure 2.

 

HTF migration was assayed by a modification of the Boyden chamber method. Five randomly chosen fields were counted at 100× magnification with a phase-contrast microscope. Results are expressed as the percentage of control cells. Experiments were performed in triplicate and repeated at least three times. HTFs of the same passage number incubated with 5‰ (vol/vol) ethanol without addition of APCs served as the control.

Figure 3.

 

HTF-mediated contraction was measured by a collagen gel contraction assay. Gel contraction of either APC-treated or untreated HTFs is expressed as the percentage of collagen gel only. HTFs were treated after polymerization with one APC per well at its IC₅₀, as determined previously (Fig. 1) . HTFs of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control. Data are expressed as the mean of results in five experiments, each performed in triplicate. Error bars, SEM.

Figure 4.

 

PKC activity was measured with a radioactive assay. HTFs were treated with one APC per dish at the IC₅₀ determined previously (Fig. 1) . HTFs of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control. Control PKC activity was set to 100%. Data are the mean of results in three experiments, each performed in duplicate. Error bars, SEM.

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