IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2006;47:364-370.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0657
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eibl, K. H.
Right arrow Articles by Welge-Lussen, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eibl, K. H.
Right arrow Articles by Welge-Lussen, U.

Inhibition of Human Retinal Pigment Epithelial Cell Attachment, Spreading, and Migration by Alkylphosphocholines

Kirsten H. Eibl, Daniel Kook, Siegfried Priglinger, Christos Haritoglou, Alice Yu, Anselm Kampik, and Ulrich Welge-Lussen

From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the effect of alkylphosphocholines (APCs) on human retinal pigment epithelium (RPE) attachment, spreading, migration, and microfilament assembly in vitro.

METHODS. Cultured RPE cells of five human donors were treated with one of four APCs (C18:1-PC, C20:1-PC, C21:1-PC, or C22:1-PC) in the presence of fetal calf serum. Cell viability was tested by the trypan blue exclusion assay. Attachment was assessed after a 2-hour incubation of RPE cells on coated 96-well-plates and subsequent MTT testing. Cellular spreading is characterized by cytoplasmic halo formation and was quantified by counting four separate fields of RPE cells allowed to spread on coated 24-well plates for 4 hours. Migration was measured by a modification of the Boyden chamber method in microchemotaxis chambers with polycarbonated filters. Microfilament assembly was assessed by immunofluorescence analysis after incubation with rhodamine-phalloidin.

RESULTS. All four APCs inhibited RPE cell attachment by more than 70% of their IC50 (C18:1-PC: 30 µM; C20:1-PC: 10 µM; C21:1-PC: 10 µM; and C22:1-PC: 10 µM). Also, APCs inhibited RPE cell spreading by more than 80% and migration by more than 90% at similar concentrations. Trypan blue staining revealed a toxicity within control limits within the concentration interval tested. Microfilament organization was significantly disturbed after incubation of RPE cells with one of the four APCs close to its IC50.

CONCLUSIONS. APCs inhibit RPE cell attachment and spreading in vitro at nontoxic concentrations. As a possible mechanism of action, APCs disturb microfilament assembly, since they are known to interfere with protein kinase C (PKC) function. This could represent a novel method of preventing even early stages of proliferative vitreoretinal diseases like proliferative vitreoretinopathy (PVR).

Proliferative vitreoretinopathy (PVR) is a major cause of persistent reduction in visual acuity after retinal detachment or severe ocular trauma.1 2 3 Clinically, PVR is characterized by the dispersion and proliferation of retinal pigment epithelial cells in the vitreous and the subsequent formation of membranes on both retinal surfaces.4 5 The contraction of these epiretinal and subretinal membranes results in tractional retinal detachment or redetachment.

Retinal pigment epithelial (RPE) cells seem to play a major role in PVR development (e.g., in retinal membrane formation and as a contractile force resulting in complex retinal redetachment).6 7 The cascade of cellular events leading to PVR is cell mediated and stimulated by serum and/or a variety of growth factors.8 9 Initial events in this cellular cascade are attachment to the retinal surface, spreading along the surface, and migration with subsequent formation of cellular membranes.10 Later cellular events present as proliferation and cell-mediated contraction of retinal membranes resulting in redetachment of the retina.

Pharmacologic prevention of PVR membrane formation has been based chiefly on antiproliferative and anti-inflammatory agents.11 12 13 14 Although many of these substances lack efficacy and may even have toxic side effects, some of them seem to reduce PVR recurrence.11 However, no pharmacological adjuvant has been established as part of a routine treatment for PVR, as recent reviews of the current literature show.15 16

In previous in vitro studies, we have shown that alkylphosphocholines (APCs) are effective inhibitors of human RPE cell proliferation and RPE-mediated collagen contraction at nontoxic concentrations.17 APCs are a new class of pharmacological agents in routine clinical use with known antitumoral (Miltex; Zentaris GmbH, Frankfurt, Germany) and antiparasitic (Impavido; Zentaris GmbH) properties.18 19 The mode of action of APCs is mediated by the cell membrane and involves the inhibition of protein kinase C in human RPE cells17 as well as in other cellular systems in vitro.20 21 The activation of PKC seems to be an important feature, not only in the late phase of PVR, as in RPE-mediated membrane contraction,22 but also during the early cellular events of PVR development, as in cellular migration,23 adhesion, and spreading.24 In addition, PKC inhibitors have been implicated in the regulation of microfilament network organization.25 Thus, a selective inhibition of this pathway by APCs could offer a new perspective on prevention of PVR.

The purpose of this study was to test whether APCs inhibit early cellular events in PVR pathogenesis, such as attachment, spreading and migration of human RPE cells in vitro and to assess microfilament assembly in the presence of serum.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
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), as described previously.17

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. Equal volumes of ethanol without addition of APCs served as the control in all experiments performed.

Human RPE Cell Culture
RPE cells were isolated from five human donors, age 20 to 71 years, within 4 to 14 hours after death as described previously26 and obtained from the Eye Bank of the Ludwig-Maximilians University Munich. 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. The neuroretina was then carefully peeled away from the RPE layer with a fine forceps. The eye cup was rinsed with Ca2+- and Mg2+-free Hank’s balanced salt solution and treated with 0.25% trypsin (Invitrogen-Gibco, Karlsruhe, Germany) for 1 hour at 37°C. After aspiration of trypsin, the eye cup 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 Bruch’s membrane.

The cell suspension was transferred to a 50-mL flask (Falcon; Corning-Costar, Wiesbaden, Germany) containing 20 mL of DMEM supplemented with 20% FCS and maintained at 37°C and 5% carbon dioxide. All cell culture dishes used were uncoated. Epithelial origin was confirmed by immunohistochemical staining for cytokeratin using a pancytokeratin antibody (Sigma-Aldrich, Deisenhofen, Germany) as described previously (data not shown).27 The cells were tested and found 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 the third and sixth passages (passages 3–6) 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 Attachment Assay
Ninety-six well plates (Nunc, Wiesbaden, Germany) were coated with 70 µL/well fibronectin (50 µg/mL in PBS [pH 7.4]; Sigma-Aldrich) for 16 hours at 4°C. Unspecific binding was blocked by 2 mg/mL ovalbumin (Sigma-Aldrich) in PBS for 1 hour at 37°C as described previously.28

RPE cells (passages 3–6) were seeded in 96-well plates (Nunc) at a density of 1 x 106 cells per well in 1 mL DMEM, 10% FCS. APCs were added in concentrations spanning the 50% inhibitory concentration (IC50) interval between 0.01 and 50 µM, as determined in preliminary assays.17 The IC50 is defined as the concentration of a drug that produces a 50% reduction in cell number at nontoxic concentrations. After 2 hours, RPE cells were then carefully washed three times with PBS using an automated plate washer (Molecular Devices, Garching, Germany).

After the washing step, the number of attached RPE cells was determined by the tetrazolium dye-reduction assay [MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide]; Sigma-Aldrich). The MTT test was performed as described by Mosmann,29 with some modifications. In brief, 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 and carefully observed to exclude crystal formation outside of cells. Formazan crystals formed were then 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, Göttingen, Germany). The number of attached living cells correlated with the absorbance (optical density, OD) measured by the MTT at 550 nm. Results from the wells were expressed as the mean percentage of the control (control OD 0.6 at 550 nm assigned as 100%). Experiments were performed in triplicate and repeated four times. RPE cells of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control.

Cell Spreading Assay
For analysis of cell spreading, 24-well plates (Nunc) were coated with 70 µL/well fibronectin (50 µg/mL in PBS, pH 7,4; Sigma-Aldrich) for 16 hours at 4°C. Unspecific binding was blocked by 2 mg/mL ovalbumin (Sigma-Aldrich) in PBS for 1 hour at 37°C, as described previously.10 Human RPE cells (passages 3–6) were seeded at a density of 5 x 104 cells per well on the fibronectin-coated wells in 1 mL DMEM and 10% FCS. APCs were added in concentrations spanning the 50% inhibitory RPE cells, as determined previously.17 RPE cells were allowed to spread for 4 hours at 37°C and 5% CO2. After they were washed with PBS three times, cells were fixed with 4% paraformaldehyde in PBS, and then stained with Coomassie brilliant blue for 3 to 4 minutes and rinsed again with PBS. For quantification of cell spreading, four separate fields were photo-documented with a phase contrast microscope (Leica, Heidelberg, Germany). Cell spreading was characterized by the formation of a clearly defined cytoplasmic halo around the cell nucleus. RPE cells demonstrating this phenomenon were counted and the percentage of the total number of RPE cells per field was calculated. Results from the wells were expressed as the mean percentage of the control. Experiments were performed in triplicate and repeated four times. RPE cells of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control.

Cell Migration Assay
Migration was determined by a modification of the Boyden’s chamber method using microchemotaxis chambers (NeuroProbe, Gaithersburg, MD) and polycarbonate filters (Nucleopore, Karlsruhe, Germany) with a pore size of 8.0 µm.30 The filters were coated with fibronectin and placed between the chambers. The lower half of the Boyden chamber was filled with 125 µL DMEM containing 20 ng/mL human recombinant platelet-derived growth factor (PDGF-BB; PeproTech, London, UK). Human RPE cells (passages 3–6) were then suspended at a density of 5 x 105 cells/mL in DMEM and 0.5% FCS and placed in the upper half of the Boyden chamber (500 µL). APCs were added in concentrations spanning the 50% inhibitory RPE cells, as determined previously.17 The cells were incubated 6 hours at 37°C in 5% CO2. After the upper side of the filter was scraped with a cotton tip to eliminate RPE cells that had not migrated through it, the filter was removed and fixed in methanol before staining with hematoxylin. Cell number in five fields chosen randomly was determined at a magnification of x200 using a phase contrast microscope (Leica). Experiments were performed in triplicate and repeated four times. RPE cells of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control.

Immunofluorescence Analysis of Microfilaments
Microfilament organization of human RPE cells (passages 3–6) was assessed by a modification of the immunofluorescence analysis protocol with rhodamine-phalloidin (AlexaFluor 488 phalloidin A 12379; Molecular Probes, Inc.), as described elsewhere.25 Cells were seeded in chamber slides (2 x 104 cells/well in DMEM and 10% FCS). After incubation for 4 hours at 37°C in 5% CO2, the medium was aspirated and adherent cells were fixed with 3.7% paraformaldehyde in PBS for 10 minutes at 20°C. After they were washed with PBS (pH 7.4), the cells were permeabilized with 0.1% Triton X-100 for 3 minutes and blocked with 1% BSA (bovine serum albumin) in PBS. Cells were then incubated with rhodamine-phalloidin (200 U/mL) and Hoechst stain (1 µg/mL) for 20 minutes in the dark. PBS was used as the base of all solutions and intervening rinses, and incubations were performed at room temperature. After mounting (Kaiser’s Glyceringelatine; Merck, Darmstadt, Germany), slides were examined under an epifluorescence microscope with appropriate excitation and emission filters under 40x magnification. Pictures of observed fields were recorded digitally. Experiments were repeated three times. RPE cells of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control.

Statistical Evaluation
Statistical analysis was performed (SPSS V 11.0; SPSS Inc., Chicago, IL). All results are expressed as the mean ± SEM as indicated. For determination of the significance of differences an ANOVA was performed. Differences with P < 0.01 were considered statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of RPE Cell Attachment by APCs
All APCs investigated inhibited attachment of human retinal pigment epithelium cells on fibronectin-coated cell culture dishes in a dose-dependent manner in vitro (Fig. 1) . At the same concentration, however, APCs with a longer carbon chain of more than C20 were more effective than C18:1-PC. This finding correlates well with the difference in the IC50 concentration determined previously17 (Table1). Consequently, the APC with the highest IC50 of approximately 30 µM, C18:1-PC, has the lowest efficacy in the inhibition of RPE cell attachment compared with those of C20:1-PC, C21:1-PC, and C22:1-PC with an IC50 of only approximately 10 µM.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 1. Inhibition of RPE cell attachment by APCs measured by a colorimetric test (MTT). RPE cells of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control. Results was expressed as the mean percentage of the control (control OD 0.6 at 550 nm was deemed 100%). Data are the mean results of four experiments, each performed in triplicate. Error bars, SEM.

 
Oleyl-phosphocholine (C18:1-PC) displayed an inhibition of RPE cell attachment at concentrations between 0.1 and 50 µM (Fig. 1) . Cells exposed to C18:1-PC at concentrations below 0.1 µM were not significantly compromised in their ability to attach compared with the control. Close to its IC50 of 26.5 µM ± 1.3 (Table 1) , C18:1-PC inhibited attachment of more than 70% of treated RPE cells. At 10 µM, 50% of RPE cells did not attach.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Summary of the Inhibitory Effects of APCs on Early Cellular Events of PVR Development In Vitro

 
All APCs with a carbon chain length of more than C20, eicosenyl-phosphocholine (C20:1-PC) heneicosenyl-phosphocholine (C21:1-PC) and erucyl-phosphocholine (C22:1-PC), inhibited RPE cell attachment at concentrations between 0.01 and 30 µM (Fig. 1) . Close to their IC50 of approximately 10 µM, respectively, more than 70% of RPE cells were unable to attach was was shown for C18:1-PC at its IC50. However, a 50% inhibition of cell attachment was achieved for all APCs with a carbon chain length of more than 20 at a concentration of only 1 µM.

Inhibition by APCs of RPE Cell Spreading
All four APCs applied caused a significant concentration-dependent inhibition of RPE cell spreading on fibronectin-coated cell culture dishes (Fig. 2) . The data suggest a difference in efficacy of the APCs in parallel to that demonstrated for RPE cell attachment. Again, close to their IC50, all four APCs were equally effective, achieving an inhibition of RPE spreading in more than 80% of cells. The concentration necessary to induce a spreading inhibition in 50% of cells was even more than 10 times higher for C18:1-PC (10 µM) than for all other APCs applied (0.1–1 µM). This corresponds well with the findings for RPE cell attachment (Table 1) .



View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 2. Human RPE cell spreading inhibition by APCs (a). Data are the mean results of four experiments each performed in triplicate. Error bars, SEM. Phase-contrast microscope photographs of control (b; arrows: halos) and treated human RPE cells (10 µM C22:1-PC; c; arrows: cells without halos) RPE cells of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control. Magnification, x100.

 
Inhibition of RPE Cell Migration by APCs
RPE cell migration was inhibited by all four APCs applied in a dose-dependent manner (Fig. 3) . Close to their IC50, respectively, each APC inhibited migration in more than 90% of RPE cells (Table 1) . APCs with a longer carbon chain of more than C20 were again far more effective at equal concentrations than was C18:1-PC. The concentration achieving a 50% inhibition of RPE cell migration was 0.1 to 1 µM for C20:1-PC, C21:1-PC, and C22:1-PC compared with 10 µM for C18:1-PC.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 3. Human RPE cell migration inhibition by APCs. Migration was assessed by a modification of the Boyden’s chamber method. Number of cells in five fields chosen randomly was assessed at a magnification of x200 with a phase-contrast microscope. Experiments were performed in triplicate and repeated four times. RPE cells of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control.

 
Disturbance of Microfilament Dynamics by APCs
F-actin assembly in human RPE cells was significantly disturbed after incubation with each one of the four APCs applied close to its IC50 (Fig. 4c 4d 4e 4f 4g 4h 4i 4j ; C18:1-PC, 30 µM: Figs. 4c 4d ; C20:1-PC, 10 µM: Figs. 4e 4f ; C21:1-PC, 10 µM: Figs. 4g 4h ; C22:1-PC, 10 µM: Figs. 4i 4j ). Control cells displayed a well organized actin skeleton with fibers extending throughout the cytoplasm into the cell membrane (Figs. 4a 4b) .



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 4. Microfilament organization of human RPE cells (passages 3–6) was assessed by immunofluorescence analysis with rhodamine-phalloidin. Cells were seeded in chamber slides, and APCs were added close to their IC50 (Table 1) . After incubation for 4 hours, adherent cells were fixed, washed, and incubated with rhodamine-phalloidin and Hoechst stain. Slides were examined under an epifluorescence microscope with appropriate excitation and emission filters. Images of observed fields were recorded digitally. Control cells displayed an elaborate network of precisely organized F-actin filaments (a, b; arrows). The F-actin filament architecture becomes disorganized with massive disruption of filament bundles after exposure to APCs in nontoxic concentrations (c–j; arrows). Experiments were repeated three times. RPE cells of the same passage number incubated with equal volumes of ethanol without addition of APCs served as the control (a, b: control; c, d: C18:1-PC, 30 µM; e, f: C20:1-PC, 10 µM; g, h: C21:1-PC, 10 µM; i, j: C22:1-PC, 10 µM).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
On the cellular level, the development of PVR can be described as a cascade of specific cellular events within the retina and on both retinal surfaces involving the vitreous. Initial cellular events in early PVR can be defined as attachment of RPE cells on the retinal surfaces followed by cytoplasmic spreading and cellular migration along these surfaces. Later stages in PVR pathogenesis are characterized by cellular proliferation and membrane formation with subsequent cell-mediated membrane contraction and tractional redetachment of the retina. In a previous in vitro study, we could demonstrate that APCs are able to inhibit the late stages in PVR development associated with RPE proliferation and cell-mediated membrane contraction.17 In this study, APCs were found to be effective inhibitors of early cellular events in PVR pathogenesis like RPE cell attachment, spreading, and migration at nontoxic concentrations.

We are aware of the limitations of our results by using fibronectin alone as a substrate for the investigation of key cellular events in early PVR. Alternatively, we and others31 32 demonstrated that RPE cells show a clear preference for fibronectin over other provisional extracellular matrix (ECM) components such as collagen type I and laminin in early PVR.33 Therefore, we believe that the inhibition of RPE cell attachment by APCs on fibronectin is a good example for the potential of these substances. Even if APCs were insufficient in their inhibition of these key cellular events on other provisional ECM substrates, their effect on fibronectin would interfere with early PVR development significantly.

Our data suggest that the effect of APCs on the key cellular events in early PVR is due to an interference of these substances with the microfilament assembly in human RPE cells. The inhibition of PKC activity by APCs17 seems to be linked with the microfilament assembly in these cells resulting in a decreased ability of the RPE to attach, spread and migrate.23 25 Thus, the inhibition of the initial stages in PVR development could prevent a disease progression to the more advanced stages commonly associated with a severe reduction in visual acuity.

In the present in vitro study, we have shown that APCs can inhibit even early cellular events in PVR development at concentrations below the IC5017 in a dose-dependent manner in the presence of serum. The dispersion and accumulation of serum components in the vitreous cavity is known to be a crucial event for in vivo PVR development due to a breakdown of the blood–retinal barrier.34 35 Serum has been identified as a strong stimulator of RPE proliferation36 and contains fibrogenic (basic fibroblast growth factor; bFGF) and inflammatory (interleukin 6; IL-6) growth factors. These factors have recently been found to be concentrated in the retro-oil fluid after complicated retinal detachment surgery prompting the development of PVR after surgery.37 A new therapeutic approach for the treatment of PVR should therefore be able to act in the presence of these growth factors.

Other pharmacologic agents currently in discussion for PVR treatment have not been effective in the presence of serum and, moreover, are not able to influence all the cellular events mentioned. For example, the most promising combination of 5-fluorouracil (5-FU) and low-molecular-weight heparin (LMWH) has been shown to inhibit RPE proliferation only.38 Clinically, there was no statistically significant difference in visual outcome of treated versus untreated patients.34 Daunomycin, another candidate for PVR treatment, inhibits cellular proliferation and migration, but the effective dose seems to be close to the toxic one which has prevented a routine clinical application so far.39 40 Also, a combination therapy of dexamethasone and heparin has not shown statistically significant results regarding PVR recurrence with at the same time an increased incidence of postoperative vitreous hemorrhage in the treated group.41 Because of a lack of efficacy and side effects of these substances, none of them is currently in routine clinical use for the prevention and/or treatment of PVR.

In summary, our results indicate that APCs are able to inhibit not only the late stages of PVR development like RPE cell proliferation and cell-mediated membrane-contraction but also the early cellular events like RPE cell attachment, spreading and migration in a dose-dependent manner at nontoxic concentrations. As a mechanism of action, APCs seem to interfere with microfilament assembly in human RPE cells by inhibition of PKC. The first intraocular in vivo toxicity study of erucylphosphocholine in a rat model displayed no retinal toxicity even at the 10-fold IC50 when administered intravitreally.42 Therefore, APCs may become a new treatment option for prevention of PVR.


   Acknowledgements
 
The authors thank Katja Obholzer for expert technical assistance.


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

Submitted for publication May 25, 2005; revised August 31, 2005; accepted November 23, 2005.

Disclosure: K.H. Eibl, None; D. Kook, None; S. Priglinger, None; C. Haritoglou, None; A. Yu, 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.

Corresponding author: Ulrich Welge-Lussen, Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Mathildenstrasse 8, 80336 Munich, Germany; uwelge{at}med.uni-muenchen.de.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tani P, Robertson DM, Langworthy A. Prognosis for central vision and anatomic reattachment in rhegmatogenous retinal detachment with macular detachment. Am J Ophthalmol. 1981;92:611–620.[ISI][Medline][Order article via Infotrieve]
  2. Charteris DG. Proliferative vitreoretinopathy: pathobiology, surgical management, and adjunctive treatment. Br J Ophthalmol. 1994;79:953–960.
  3. Cardillo JA, Stout JT, LaBree L, et al. Post-traumatic proliferative vitreoretinopathy: the epidemiologic profile, onset, risk factors, and visual outcome. Ophthalmology. 1997;104:1166–1173.[ISI][Medline][Order article via Infotrieve]
  4. Machemer R, Laqua H. Pigment epithelium proliferation in retinal detachment (massive periretinal proliferation). Am J Ophthalmol. 1975;80:1–23.[ISI][Medline][Order article via Infotrieve]
  5. Kampik A, Kenyon KR, Michels RG, Green WR, de la Cruz ZC. Epiretinal and vitreous membranes: comparative study of 56 cases. Arch Ophthalmol. 1981;99:1445–1454.[Abstract]
  6. Hiscott P, Sheridan C, Magee RM, Grierson I. Matrix and the retinal pigment epithelium in proliferative retinal disease. Prog Retin Eye Res. 1999;18:167–190.[CrossRef][ISI][Medline][Order article via Infotrieve]
  7. Carrington L, McLeod D, Boulton M. IL-10 and antibodies to TGF-beta2 and PDGF inhibit RPE-mediated retinal contraction. Invest Ophthalmol Vis Sci. 2000;41:1210–1216.[Abstract/Free Full Text]
  8. Kon CH, Occleston NL, Aylward GW, Khaw PT. Expression of vitreous cytokines in proliferative vitreoretinopathy: a prospective study. Invest Ophthalmol Vis Sci. 1999;40:705–712.[Abstract]
  9. Hinton DR, He S, Jin ML, Barron E, Ryan SJ. Novel growth factors involved in the pathogenesis of proliferative vitreoretinopathy. Eye. 2002;16:422–428.[CrossRef][ISI][Medline][Order article via Infotrieve]
  10. Priglinger SG, Alge CS, Neubauer AS, et al. TGF-beta2-induced cell surface tissue transglutaminase increases adhesion and migration of RPE cells on fibronectin through the gelatin-binding domain. Invest Ophthalmol Vis Sci. 2004;45:955–963.[Abstract/Free Full Text]
  11. Asaria RHY, Kon CH, Bunce C, et al. Adjuvant 5-fluouracil and heparin prevents proliferative vitreoretinopathy. Ophthalmology. 2001;108:1179–1183.[CrossRef][ISI][Medline][Order article via Infotrieve]
  12. Faude F, Enzmann V, Poschmann E, Hoffmann S, Wiedemann P. R-(+)-verapamil, S-(–)-verapamil, and racemic verapamil inhibit human retinal pigment epithelial cell contraction. Graefes Arch Clin Exp Ophthalmol. 2000;238:537–541.[CrossRef][ISI][Medline][Order article via Infotrieve]
  13. Esser P, Tervooren D, Heimann K, et al. Intravitreal daunomycin induces multidrug resistance in proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci. 1998;39:164–170.[Abstract/Free Full Text]
  14. Yang CS, Khawly JA, Hainsworth DP, et al. An intravitreal sustained-release triamcinolone and 5-fluorouracil codrug in the treatment of experimental proliferative vitreoretinopathy. Arch Ophthalmol. 1998;116:69–77.[Abstract/Free Full Text]
  15. Kirchhof B. Strategies to influence PVR development. Graefes Arch Clin Exp Ophthalmol. 2004;242:699–703.[ISI][Medline][Order article via Infotrieve]
  16. Scheer S, Morel C, Touzeau O, Sahel JA, Laroche L. Pharmacological adjuvants for surgical treatment of proliferative vitreoretinopathy. J Fr Ophtalmol. 2004;27:1051–1059.[ISI][Medline][Order article via Infotrieve]
  17. Eibl KH, Banas B, Schoenfeld CL, et al. Alkylphosphocholines inhibit proliferation of human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2003;44:3556–3561.[Abstract/Free Full Text]
  18. Leonard R, Hardy J, van Tienhoven G, et al. Randomized, double-blind, placebo-controlled, multicenter trial of 6% miltefosine solution, a topical chemotherapy in cutaneous metastases from breast cancer. J Clin Oncol. 2001;21:4150–4159.
  19. Sundar S, Jha TK, Thakur CP, et al. Oral miltefoesine for Indian visceral leishmaniasis. N Engl J Med. 2002;347:1739–1746.[Abstract/Free Full Text]
  20. Überall F, Oberhuber H, Maly K, Zaknun J, Demuth L, Grunicke HH. Hexadecylphosphocholine inhibits inositol phosphate formation and protein kinase C activity. Cancer Res. 1991;51:807–812.[Abstract/Free Full Text]
  21. Shoji M, Raynor RL, Fleer EAM, Eibl H, Vogler WR, Kuo JF. Effects of hexadecylphosphocholine on protein kinase C and TPA-induced differentiation of HL60 cells. Lipids. 1991;26:145–149.[ISI][Medline][Order article via Infotrieve]
  22. Sakamoto T, Hinton DR, Sakamoto H, et al. Collagen gel contraction induced by retinal pigment epithelial cells and choroidal fibroblasts involves the protein kinase C pathway. Curr Eye Res. 1994;13:451–459.[ISI][Medline][Order article via Infotrieve]
  23. Murphy TL, Sakamoto T, Hinton DR, et al. Migration of retinal pigment epithelium cells in vitro is regulated by protein kinase C. Exp Eye Res. 1995;60:683–695.[CrossRef][ISI][Medline][Order article via Infotrieve]
  24. Goel HL, Dey CS. Role of protein kinase C during insulin mediated skeletal muscle cell spreading. J Muscle Res Cell Motil. 2002;23:269–277.[CrossRef][ISI][Medline][Order article via Infotrieve]
  25. Masson-Gadais B, Salers P, Bongrand P, Lissitzky J-C. PKC regulation of microfilament network organization in keratinocytes defined by a pharmacological study with PKC activators and inhibitors. Exp Cell Res. 1997;236:238–247.[CrossRef][ISI][Medline][Order article via Infotrieve]
  26. Edwards RB. Culture of mammalian retinal pigment epithelium and neural retina. Methods Enzymol. 1982;81:39–43.[ISI][Medline][Order article via Infotrieve]
  27. Robey HL, Hiscott PS, Grierson I. Cytokeratins and retinal pigment epithelial behaviour. J Cell Sci. ;102:329–340.
  28. Lotz MM, Burdsal CA, Erickson HP, McClay DR. Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response. J Cell Biol. 1989;109:1795–1805.[Abstract/Free Full Text]
  29. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–56.[CrossRef][ISI][Medline][Order article via Infotrieve]
  30. Figari IS, Mori NA, Palladino MA, Jr. Regulation of neutrophil migration and superoxide production by recombinant tumor necrosis factors-alpha and -beta: comparison to recombinant interferon-gamma and interleukin-1 alpha. Blood. 1989;70:979–984.
  31. Wagner M, Benson MT, Rennie IG, MacNeil C. Effects of pharmacological modulation of intracellular signalling systems on retinal pigment epithelial cell attachment to extracellular matrix proteins. Curr Eye Res. 1995;14:373–384.[ISI][Medline][Order article via Infotrieve]
  32. Priglinger SG, Alge CS, Neubauer AS, et al. TGF-beta-2 induced cell surface tissue transglutaminase increases adhesion and migration of RPE cells on fibronectin through the gelatin-binding domain. Invest Ophthalmol Vis Sci. 2004;45:955–963.[Abstract/Free Full Text]
  33. Scheiffarth OF, Kampik A, Gunther H, von der Mark K. Proteins of the extracellular matrix in vitreoretinal membranes. Graefes Arch Clin Exp Ophthalmol. 1988;226:357–361.[CrossRef][ISI][Medline][Order article via Infotrieve]
  34. Campochiaro PA, Bryan JA, III, Conway BP, Jaccoma EH. Intravitreal chemotactic and mitogenic activity: implication of blood-retinal barrier breakdown. Arch Ophthalmol. 1986;104:1685–1687.[Abstract]
  35. Martini B, Pandey R, Ogden TE, Ryan SJ. Cultures of human retinal pigment epithelium: modulation of extracellular matrix. Invest Ophthalmol Vis Sci. 1992;33:516–521.[Abstract/Free Full Text]
  36. Hecquet C, Lefevre G, Valtink M, Engelmann K, Mascarelli F. Activation and role of MAP kinase-dependent pathways in retinal pigment epithelium cells: JNK1, P38 kinase, and cell death. Invest Ophthalmol Vis Sci. 2003;44:1320–1329.[Abstract/Free Full Text]
  37. Asaria RHY, Kon CH, Bunce C, et al. Silicone oil concentrates fibrogenic growth factors in the retro-oil fluid. Br J Ophthalmol. 2004;88:1439–1442.[Abstract/Free Full Text]
  38. Stern WH, Lewis GP, Erickson PA. Fluorouracil therapy for proliferative vitreoretinopathy after vitrectomy. Am J Ophthalmol. 1983;96:43–51.[ISI][Medline][Order article via Infotrieve]
  39. Santana M, Wiedemann P, Kirmani M. Daunomycin in the treatment of experimental proliferative vitreoretinopathy: retinal toxicity of intravitreal daunomycin in the rabbit. Graefes Arch Clin Exp Ophthalmol. 1984;221:210–213.[CrossRef][ISI][Medline][Order article via Infotrieve]
  40. Steinhorst UH, Hatchell DL, Chen EP, Machemer R. Ocular toxicity of daunomycin: effects of subdivised doses on the rabbit retina after vitreous gas compression. Graefes Arch Clin Exp Ophthalmol. 1993;231:591–594.[CrossRef][ISI][Medline][Order article via Infotrieve]
  41. Williams RG, Chang S, Comaratta MR. Does the presence of heparin and dexamethasone in the vitrectomy infusate reduce proliferation in proliferative vitreoretinopathy?. Graefes Arch Clin Exp Ophthalmol. 1996;234:496–503.[ISI][Medline][Order article via Infotrieve]
  42. Schuettauf F, Eibl KH, Thaler S, et al. Toxicity study of erucylphosphocholine in the rat. Curr Eye Res. 2005;30:813–820.[CrossRef][ISI][Medline][Order article via Infotrieve]



This article has been cited by other articles:


Home page
 IOVSHome page
K. H. Eibl, G. P. Lewis, K. Betts, K. A. Linberg, A. Gandorfer, A. Kampik, and S. K. Fisher
The Effect of Alkylphosphocholines on Intraretinal Proliferation Initiated by Experimental Retinal Detachment
Invest. Ophthalmol. Vis. Sci., March 1, 2007; 48(3): 1305 - 1311.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eibl, K. H.
Right arrow Articles by Welge-Lussen, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eibl, K. H.
Right arrow Articles by Welge-Lussen, U.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS