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(Investigative Ophthalmology and Visual Science. 2002;43:483-489.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

PI3K and PLC{gamma} Play a Central Role in Experimental PVR

Yasushi Ikuno, Fee-Lai Leong and Andrius Kazlauskas

From The Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. It has been reported that the platelet-derived growth factor (PDGF)-{alpha} receptor ({alpha}PDGFR) is required for experimental proliferative vitreoretinopathy (PVR) in rabbits. This study investigated which of the signaling enzymes downstream of the {alpha}PDGFR participate in PVR.

METHODS. A panel of cell lines that expressed {alpha}PDGFR signaling mutants were made and characterized. These cell lines were used in a rabbit model of PVR and in an in vitro collagen type I contraction assay.

RESULTS. Phosphoinositide 3-kinase (PI3K) and, to a lesser extent, phospholipase C (PLC)-{gamma} were the signaling enzymes required for the {alpha}PDGFR to mediate PVR. Furthermore, the cells lines that were the most effective at inducing PVR displayed the most potent activity in the in vitro contraction assay.

CONCLUSIONS. PI3K and PLC{gamma} are necessary downstream effectors of the {alpha}PDGFR in experimental PVR. Consequently, these two signaling enzymes are required for one or more of the cellular responses (chemotaxis, proliferation, extracellular matrix production, contraction) that contribute to PVR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Proliferative vitreoretinopathy (PVR) occurs in up to 10% of patients undergoing surgery to reattach the retina.1 Approximately 60% to 80% of the patients that undergo repeated surgery achieve anatomic success.2 3

There are numerous studies indicating that growth factors contribute to PVR.4 5 6 7 8 9 10 11 12 13 14 Using a simple rabbit model of the disease, we have found that platelet-derived growth factor (PDGF)-{alpha} receptor ({alpha}PDGFR) plays a major role in PVR.4 Unlike the PDGF ß receptor, which is activated by a small subset of the PDGF family, the {alpha}PDGFR is activated by many PDGF isoforms, including PDGF-AA, -AB, -BB, and -CC.15 16 17 Ligand binding activates the receptor, whereupon it becomes tyrosine phosphorylated and associates with a variety of SH2 domain–containing signaling enzymes.18 These include Src family kinases, the phosphotyrosine phosphatase SHP-2, phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC)-{gamma}. These signaling enzymes are required to mediate PDGF-dependent cellular responses, and different pathways seem to be involved in the distinct cellular responses. For instance, PI3K is required to drive cells into the S phase, whereas the combination of Src family kinases, PI3K, and PLC{gamma} are necessary for PDGF-dependent chemotaxis.19 The Src family kinases are cytosolic tyrosine kinases, whereas PLC{gamma} is a phospholipase that cleaves phosphatidylinositol-4,5-bis phosphate to generate diacylglycerol and inositol trisphosphate, which are potent second messengers.20 PI3K is a family of enzymes that are best known for their ability to phosphorylate phospholipids, which engage numerous other enzymes and signaling cascades.21

The PDGFR is particularly well suited to the investigation of signaling cascades that contribute to PDGF-dependent responses. This is because at least some of the signaling enzymes that are used by the PDGFR are activated after phosphorylation of the PDGFR at specific tyrosine residues. Mutating individual tyrosine phosphorylation sites selectively prevents activation of a given branch of the signaling cascade. Hence, a panel of PDGFR phosphorylation site mutants has been used to determine the relative contribution of signaling enzymes to cellular responses such as proliferation, migration, and protection from apoptosis.19 22 23 24 Mice expressing various PDGFR phosphorylation site mutants have recently been generated and used to evaluate the role of signaling enzymes in development and disease.25 26

One of the pathologic events in PVR is the contraction of the epiretinal membrane, which results in tractional retinal detachment (TRD).2 Contraction of cells within the epiretinal membrane is commonly believed to cause TRD. A large fraction of this epiretinal membrane is composed of extracellular matrix (ECM) proteins, such as type I collagen.27 The contraction event appears to require binding of cellular integrins, such as {alpha}2ß1, to the ECM, in that administration of peptides that interfere with this interaction prevent PVR.28 29 30 31

In this study we investigated which of the known {alpha}PDGFR effectors is required for PVR and for cellular contraction. Using a panel of cell lines that express {alpha}PDGFR-signaling mutants, we found that PI3K and, to a lesser extent, PLC{gamma} are required for both contraction and PVR. Furthermore, in vitro contraction correlates well with in vivo PVR, suggesting that cellular contraction is an important component of PVR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
F cells are a simian virus (SV)-40 immortalized line of mouse embryo fibroblasts derived from mice nullizygous for both the {alpha} and ß PDGFRs. They were generously provided by Michelle Tallquist and Philippe Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA). The cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS); the serum concentration was reduced to 1% when the cells were serum starved.

The {alpha}PDGFRs were expressed in F cells, by using a retroviral system.19 32 33 Briefly, wild-type (WT) or mutated PDGFR cDNA was subcloned into the retroviral vector pLHDCX2 . The cDNA constructs were transfected into the virus–producing 293 GPG cell line, using a reagent for transformation of eukaryotic cells (Lipofectamine; Life Technologies, Gaithersburg, MD). The virus-containing supernatant was collected for 5 days and then concentrated by centrifugation at 25,000g at 4°C for 90 minutes. The virus was resuspended in a small volume overnight and frozen at -70°C until use. F cells were infected with the appropriate retrovirus in the presence of 4 µg/mL Polybrene (Sigma Chemical Co., St. Louis, MO) overnight and then selected by growth in medium containing 5 mM histidinol. In all cases, mass populations of drug-resistant cells were used. In Figure 3 the CX2 cells were generated by infecting F cells with an empty-expression vector and selecting by growth in medium containing 5 mM histidinol.



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  		Figure 3. Identification of signaling enzymes that are required for {alpha}PDGFR-mediated contraction. Cells expressing the indicated {alpha}PDGFRs were subjected to the in vitro collagen gel assay in the presence of buffer (A), 10% FBS (B), or 50 ng/mL PDGF-BB (C). The data presented are the mean ± SD measured at the 48-hour time point. Each experimental condition was assayed in triplicate, and at least three independent experiments were performed. Cells that have no PDGFRs (CX2) or express a kinase-inactive receptor (R627) failed to respond to PDGF. Similarly, receptors that fail to recruit multiple signaling enzymes (F7) responded poorly. PDGF-dependent contraction was either eliminated or reduced when receptors were unable to engage PI3K (F31/42) or PLC{gamma} (F1018), respectively. In contrast, the inability to recruit Src family kinases (F72/74) or SHP-2 did not markedly compromise PDGF-dependent contraction. **Significant difference (P < 0.01 by unpaired t-test) compared with the response of the WT cells.

 
{alpha}PDGFR Mutagenesis and Expression
The {alpha}PDGFR mutants were generated using site-directed mutagenesis, as previously described.19 The nature of each of the mutants and their capacity to recruit signaling enzymes are described in Table 1 .


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  		Table 1. Properties of {alpha}PDGFR Mutants

 
Immunoprecipitation and Immunoblotting
Cells were grown to 80% confluence and then starved with DMEM containing 1% FBS for 20 hours. To activate the receptors, the cells were exposed to 50 ng/mL PDGF-BB for 5 minutes. After treatment, the cells were washed twice with 20 mM HEPES (pH 7.4), 150 mM NaCl (H/S) and then lysed in EB (10 mM Tris-HCl [pH 7.4], 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 0.1% BSA, 20 µg/mL aprotinin, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride [PMSF]). Lysates were centrifuged for 15 minutes at 13,000g, the pellet was discarded, and the soluble fraction was used as the total cell lysate. The protein concentration was measured using a protein assay kit (Pierce Chemical Co., Rockford, IL), according to the manufacturer’s instructions. Receptors were immunoprecipitated from the soluble fraction with the 27P antibody (described below). Immune complexes were bound to formalin-fixed membranes of Staphylococcus aureus, spun through an EB sucrose gradient, and washed twice with EB, then with PAN (10 mM piperazine-N-N'-bis(2-ethanesulfonic acid [PIPES; pH 7.0], 100 mM NaCl, and 1% aprotinin) with 0.5% Nonidet P [NP]-40), and the final two washes were with PAN. Total cell lysates containing 20 µg protein or receptor immunoprecipitates from 1.0 x 106 cells were resolved on a 7.5% SDS-PAGE gel under reducing conditions. Proteins were transferred onto nitrocellulose membranes (Immobilon; Millipore, Bedford, MA), and the membranes were blocked using blocking reagents: either Block (10 mM Tris-HCl, [pH 7.5], 1.5 M Tris base, 150 mM NaCl, 10 mg/mL BSA, 10 mg/mL ovalbumin, and 0.05% Tween 20; Pierce Chemical Co.) for anti-phosphotyrosine antibodies, or Blotto (10 mM Tris-HCl [pH 7.5], 1.5 M Tris base, 150 mM NaCl, 10 mg/mL nonfat dry milk, and 0.05% Tween 20; Santa Cruz Biotechnology, Santa Cruz, CA) for other antibodies. The membranes were incubated with primary antibodies for 1 hour at room temperature, and washed five times with Western rinse solution (150 mM NaCl, 10 mM Tris-HCl [pH 7.5], and 1.5 mM Tris base). Afterward, they were incubated with secondary antibody for 1 hour at room temperature and washed five times with Western rinse, and the signal was developed using the enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech, Piscataway, NJ). Multiple exposures of each were obtained, and the data presented were within the linear range of the film.

Reagents and Antibodies
Recombinant human PDGF-BB was purchased from R&D Systems (Minneapolis, MN). The 27P (anti-{alpha}PDGFR), 80.8 (anti-{alpha}PDGFR), and 69.3 (anti-Ras guanosine triphosphate [GTP]–activating protein [RasGAP]) rabbit crude antisera have been described and characterized previously.35 36 Crude rabbit anti-p85 antibody was kindly provided by Alex Toker (Beth Israel Hospital, Harvard Medical School, Boston, MA). 4G10 and PY20 mouse monoclonal anti-phosphotyrosine antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY) and Transduction Laboratories (San Diego, CA), respectively. The monoclonal antibodies against PLC{gamma} and SHP-2 were purchased from Upstate Biotechnology Inc. For Western blot analysis the following dilutions were used for each primary antibodies: anti-{alpha}PDGFR, a 1:1 mixture of the 27P and 80.8 antibodies, 1:1000; anti-phosphotyrosine, 4G10:PY20 (1:1), 1:5000; 69.3, 1:4000; anti-PLC{gamma}, 1:1000; and anti-SHP-2, 1:1000. Secondary antibodies were horseradish peroxidase–conjugated anti-rabbit (catalog no. NA934; Amersham Pharmacia Biotech) or anti-mouse (catalog no. NA931; Amersham Pharmacia Biotech) antibodies diluted 1:5000.

PI3K Assay
The PI3K assay was performed as previously described.37 Briefly, immunoprecipitated {alpha}PDGFR from approximately 5 x 105 cells were incubated with phosphatidylinositol in the presence of [32P]-{gamma}-adenosine triphosphate (ATP). The reactions were terminated, and the phospholipids were extracted and resolved by thin-layer chromatography. The radioactive product of the reaction (phosphoinositide-3-phosphate) was detected by autoradiography.

Collagen I Contraction Assay
The contraction assay was performed as previously described,38 with slight modifications. Briefly, cells were suspended in 1.5 mg/mL neutralized collagen I (Cohesion Vitrogen 100, Palo Alto, CA) at a density of 106 cells/mL and placed in a 24-well plate (Falcon, Franklin Lakes, NJ) that had been preincubated for 12 to 16 hours with 5 mg/mL BSA in phosphate-buffered saline (PBS). The gel was solidified by incubating at 37°C for 90 minutes, and the wells were flooded with DMEM and 5 mg/mL BSA supplemented with the agent to be tested. The gels were incubated at 37°C with 5% CO2. The media were replaced every 24 hours, and the gel diameter was measured after 24, 48, and 72 hours. The extent of contraction was calculated by subtracting the diameter of the well at a given time point from the initial diameter (15 mm). Each experimental condition was assayed in triplicate, and at least three independent experiments were performed.

Rabbit Experimental PVR Model
PVR was induced in rabbit eyes according to a method previously described.39 Briefly, rabbits were anesthetized and subjected to gas vitrectomy by injection of 0.4 mL of expanding perfluoropropane gas (C3F8) into the vitreous cavity. Three days later, 1 x 105 cells were suspended in 0.1 mL DMEM and coinjected into the vitreous with 0.1 mL platelet-rich plasma. A single investigator evaluated the retinal status in an unmasked fashion using an indirect ophthalmoscope at 1, 4, 7, 14, 21, and 28 days after cell injection. PVR grading was according to the method of Fastenberg et al.,40 41 as follows: 0, no abnormality; 1, vitreous strand; 2, traction of the retina; 3, retinal detachment involving less than two quadrants; 4, extended retinal detachment including more than two quadrants; and 5, total retinal detachment. All procedures involving the animals were performed under aseptic conditions, pursuant to the regulations of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Statistic Analysis
An unpaired t-test was performed to detect statistical differences in the contraction assay; a nonparametric Mann-Whitney test was used for animal PVR experiments. In all cases, P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of {alpha}PDGFR Mutants
The first step of this project was to create and characterize a panel of cell lines that express {alpha}PDGFR mutants, which selectively fail to associate with signaling enzymes (Table 1) . We used F cells, which is a mouse embryo fibroblast cell line devoid of any PDGFR proteins, because they are derived from embryos that have neither of the PDGFR genes. Their having no endogenous PDGFRs makes these cells uniquely suited for experiments using PDGFR mutants, because endogenous PDGFRs activate the complete set of signaling enzymes and hence negate the selective engagement of a subset of signaling pathways made possible by the use of receptor mutants. An additional feature of these cells is that the parental line induces PVR poorly when injected into rabbits, and re-expression of the {alpha}PDGFR greatly increases their PVR potential.4 The mutant {alpha}PDGFRs were stably expressed in F cells using a retroviral expression system. Western blot analysis of the resultant cell lines indicated that each of the mutants were expressed to within twofold of the level of the WT receptor (Fig. 1A ). The mature form of the receptor is glycosylated (Fig. 1A , arrow) and runs as a smear, whereas the immature, unglycosylated receptor is the better-resolved lower molecular weight species.



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  		Figure 1. Characterization of F cells expressing {alpha}PDGFR mutants. (A) Western blot analysis of total cell lysates. Cells were grown to 80% confluence, serum starved overnight, and lysed. Twenty micrograms of protein was subjected to Western blot analysis, using an antibody specific for either {alpha}PDGFR (top) or RasGAP, which was included as a control for the amount of protein loaded (bottom). Arrow: glycosylated mature form of the receptor (180 kDa), whereas the 160-kDa species is the immature form of the receptor. RasGAP is 120 kDa. Multiple exposures of each of the blots in (A) and (B) were obtained, and the results presented were within the linear range of the film. (B) PDGF-dependent tyrosine phosphorylation and recruitment of signaling enzymes. F cells expressing various {alpha}PDGFR mutants were grown to 80% confluence and serum starved overnight. Cells were either left resting (-) or stimulated with 50 ng/mL PDGF-BB (+) for 5 minutes and then lysed. The {alpha}PDGFR was immunoprecipitated with an {alpha}PDGFR antibody, and the resultant immunoprecipitates were resolved by 7.5% SDS-PAGE and transferred onto nitrocellulose membrane. The segment of the blot containing the {alpha}PDGFR was first blotted with anti-phosphotyrosine antibodies and then stripped and reprobed with an anti-{alpha}PDGFR antibody (top). Portions of the membrane that contained the proteins of the appropriate molecular mass were immunoblotted for PLC{gamma} (148 kDa), the regulatory subunit of PI3K (p85, 85 kDa), or SHP-2 (64 kDa). (C) Receptor-associated PI3K activity. Aliquots of the immunoprecipitates used in (B) were subjected to a PI3K activity assay. This assay detects PI3K that has coprecipitated with the {alpha}PDGFR. Immunoprecipitates were incubated with phosphatidylinositol and [32P]-{gamma}ATP, the reaction was stopped, and the phospholipids were extracted and separated by chromatography. The resultant chromatogram was exposed to x-ray film to detect radioactive phosphatidylinositol phosphate (PIP). IB, immunoblot; the antibodies used for the immunoblot assays are indicated to the right; P-Y, anti-phosphotyrosine.

 
To characterize the {alpha}PDGFR mutants, we examined their ability to undergo ligand-dependent tyrosine phosphorylation and to associate with signaling enzymes. The top panels of Figure 1B show that all the receptors, with the exception of the kinase-inactive mutant (R627) were tyrosine phosphorylated after exposure of the cells to PDGF. Seven tyrosine phosphorylation sites are missing in the F7 receptor, and this is probably why this receptor is less phosphorylated. The WT {alpha}PDGFR coprecipitated with PLC{gamma}, p85 and SHP-2, whereas the F7 and R627 receptors failed to recruit any of these signaling enzymes (Fig. 1B , bottom panels). The F720, F31/42, and F1018 receptors displayed a more selective defect, so that they failed to efficiently associate with SHP-2, p85, and PLC{gamma}, respectively. To measure the PI3K activity that coprecipitated with the PDGFRs, the receptor immunoprecipitates were subjected to an in vitro PI3K assay. Consistent with the findings of the p85 Western blot (Fig. 1B) , PI3K activity coprecipitated with the WT receptor, and the amount of activity was greatly enhanced by PDGF stimulation (Fig. 1C) . In contrast, PI3K activity did not detectably coprecipitate with the F31/42 receptor isolated from either resting or PDGF-stimulated cells (Fig. 1C) . We have previously characterized the F72/74 receptor expressed in these cells, and it selectively failed to associate with Src family kinases.42 Finally, the behavior of the {alpha}PDGFR mutants in F cells is very similar to our previous observation when the receptor was expressed in a different fibroblast cell line (Ph cells).19 In summary, this panel of {alpha}PDGFR mutants is suitable for assessing the importance of several parameters of receptor function. These include kinase activity, global ability to recruit signaling enzymes, and the importance of specific signaling pathways that are initiated by PI3K, PLC{gamma}, SHP-2, or the Src family kinases.

Involvement of the PI3K and PLC{gamma} Pathways in Experimental PVR
To identify the signaling enzymes that participate in PVR, we assayed cells expressing the various {alpha}PDGFR mutants in a rabbit PVR model. In this model, rabbits first undergo gas vitrectomy, and then cells are coinjected with platelet-rich plasma. The formation of an epiretinal membrane, retinal traction, and retinal detachment is observed in living animals over 28 days. By day 14, clear differences were noted between the groups of rabbits, and these differences persisted and intensified over the following 2 weeks (Fig. 2 , Table 2 ). By day 28, extensive retinal detachment (score of 3 or greater) was observed in most of the animals in the WT and F720 groups, whereas the disease was markedly less severe in the R627, F7, and F31/42 groups (Fig. 2 , Table 2 ). Most of the animals in these latter groups had no retinal detachment. Instead, they remained disease free (score of 0), showed development of a membrane (score of 1), or showed a membrane with tractional force (score of 2). This very modest PVR response was comparable to that seen when rabbits were injected with the parental cells, which have no PDGFRs.4 Although the response of the F1018 group was quite heterogeneous, there was a statistically significant difference between the mean score of the WT and F1018 groups (Fig. 2 , Table 2 ). We have found that cells expressing the F72/74 {alpha}PDGFR induces PVR with slightly accelerated kinetics.42 Because this mutant fails to activate Src family kinases, it appears that this class of signaling enzymes modestly attenuates PVR. Taken together, the data in Figure 2 and Table 2 indicate that the kinase activity of the {alpha}PDGFR and its ability to engage signaling pathways such as PI3K and PLC{gamma} are critical components of the signaling cascades that drive PVR.



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  		Figure 2. Identification of signaling enzymes that are required for PVR. PVR was induced by injecting F cells expressing the indicated {alpha}PDGFR into the vitreous of the rabbit eyes. The PVR score was evaluated on days 1, 4, 7, 14, 21, and 28 after surgery. The data for days 14 and 28 are presented in (A) and (B), respectively. (•) Score for individual rabbits; at the bottom of each column is the percentage of animals that had retinal detachments (graded 3 or higher). The response to parental F cells was previously reported4 and was the same as the R627, F7, or F31/42 groups. There was a statistically significant difference in the median value between the WT and all other groups, with the exception of the F720 group. Details of the statistical analysis are presented in Table 2 . A description of each of the receptors is in Table 1 ; the numbers in parentheses indicate the number of rabbits in each group.

 

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  		Table 2. The PVR Potential of Cells Expressing the {alpha}PDGFR Mutants

 
Involvement of the PI3K and PLC{gamma} Pathways in PDGF-Dependent Contraction
An important component of PVR is the tractional force generated when cells of the epiretinal membrane contract. To investigate the signaling pathways that participate in contraction, we subjected each of the cells in the panel to an in vitro collagen gel contraction assay. The cells expressing the WT receptor contracted when PDGF was added to the medium, whereas no contraction was seen in cells expressing the kinase-inactive (R627) receptor or the receptor unable to recruit signaling enzymes (F7; Fig. 3C ). The response of all three cell types was comparable under the negative (buffer) and positive (10% FBS) control conditions (Figs. 3A 3B , respectively). Thus, contraction required that the receptor have kinase activity and be able to recruit signaling enzymes.

Analysis of the contraction response of the other members of the panel revealed which of the signaling enzymes was most important. A complete loss of the contraction response was observed in cells that expressed receptors unable to recruit PI3K (F31/42) (Fig. 3C) . This observation is consistent with previous reports showing that PDGF-dependent contraction is blocked when cells are treated with inhibitors of PI3K, or express PDGFR mutants unable to associate with PI3K.25 43 Failure to engage PLC{gamma} (in the case of the F1018 receptor) also reduced the ability of the receptor to promote contraction, although not as severely as the loss of PI3K (Fig. 3C) . In contrast, eliminating the contribution of PDGF-driven activation of Src family kinases (F72/74 receptor) had no effect on contraction (Fig. 3C) . The cells expressing the receptor that did not recruit SHP-2 (F720) displayed a somewhat reduced response (Fig. 3C) , but this was not routinely observed. We conclude that PDGF-dependent contraction requires that the {alpha}PDGFR be kinase active and be able to engage PI3K and, to a lesser extent, PLC{gamma}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used a novel system to identify signaling enzymes that participate in PVR. Our results indicate that PI3K and, to a lesser extent, PLC{gamma} are essential for PVR in a rabbit model of the disease.

Because all the retinal evaluations were performed by a single investigator in an unmasked fashion, it is possible that the results contain some degree of bias. The major conclusion of the study is that only a subset of the experimental groups (those injected with cells expressing the WT or F720 {alpha}PDGFR) developed extensive retinal detachments. The retinal status of these animals was easily distinguished from the other groups that formed only membranes or membranes and retinal traction. The large differences between the two types of outcomes reduce the possibility of bias in these experiments.

Is it possible to relate the findings obtained with this animal model of PVR to the clinical setting? As with all models, this is not a perfect recreation of the disease that afflicts humans, and perhaps the most obvious difference is the cell type used to induce PVR. Whereas retinal pigment epithelial cells are commonly believed to be the predominant cell type of the epiretinal membrane isolated from humans,2 3 44 45 we used a mouse embryo fibroblast cell line. Although the use of RPE cells would have better approximated the disease, it is not possible to use receptor mutants in cells that naturally express the PDGFR, because the endogenous receptor activates all signaling pathways and hence would compensate for the signaling defect of mutant receptors. These signaling mutants are useful only in cells that have no endogenous PDGFRs. For this reason we chose the F cells, which are isolated from embryos that have both of the PDGFR genes eliminated by gene targeting. Unfortunately, the {alpha}PDGFR is required for mouse development,46 47 and the double-knockout animals die at midgestation4 ; therefore, it does not seem to be possible to isolate RPE cells with the appropriate genetic background at the present time. Although the F cells enabled us to use the panel of PDGFR mutants, additional studies are needed to evaluate the clinical relevance of the novel and exciting information that has emerged using this particular PVR model.

How do PI3K and PLC{gamma} participate in the events leading to PVR in this model? One possibility is that these signaling enzymes mediate cellular responses that are essential for the development of PVR. Indeed, we have found that PI3K is one of the effectors of {alpha}PDGFR-dependent cell cycle progression, chemotaxis,19 and contraction (Fig. 3) . Similarly, PLC{gamma} is required for chemotaxis19 and contraction (Fig. 3) . These experimental results support the commonly held belief that cell proliferation, migration, and contraction are essential cellular responses that contribute to PVR. It is also possible that PI3K and PLC{gamma} are required for some essential process in disease progression beyond these cellular events. For instance, the synthesis and organization of an extracellular matrix is likely to be central to PVR, because the bulk of an epiretinal membrane is ECM.27

An additional benefit of knowing which signaling enzymes are essential for PVR is that inhibitors of these signaling enzymes become potential therapeutic agents to prevent and/or treat PVR. This is particularly exciting in the case of PI3K, the most important signaling enzyme in PVR, because several well-characterized inhibitors already exist, and there is a sizable effort to develop additional agents that selectively and effectively block the activity of PI3K.

The development of a panel of cell lines that display a range of responses in a contraction assay has enabled us to assess the importance of this cellular parameter in PVR. Comparison of the data in Figures 2 and 3 indicates that those receptors that fail to promote contraction of collagen gels in the in vitro assay were the ones with low PVR potential. Similarly, the receptors that have high PVR potential were the ones that stimulated the best response in the in vitro contraction assay. This suggests that the ability of cells to contract is an important component of the disease. Contraction is commonly believed to involve the interaction between integrins and ECM.48 Consequently, it is possible that signaling enzymes such as PI3K and/or PLC{gamma} are involved with upregulation of integrins or the secretion of ECM. Alternatively, these signaling enzymes may play a role in regulating enzymes such as myosin light chain kinase that mediate the intracellular events responsible for the mechanics of the contraction event.38 49 50 51 52 Additional experiments are needed to investigate these possible scenarios, and to further evaluate the relative importance of cellular contraction in the development of PVR.

There is a pressing need for agents that effectively prevent PVR. To this end, in vitro–based assays are much more amenable to large-scale screening of libraries than in vivo assays. In vitro assays for cell proliferation, migration, and contractions are all well established and can be used individually or in combination to identify candidates that can be subsequently evaluated for their potential to block PVR in the in vivo setting.


   Acknowledgements
 
The authors thank the members of Kazlauskas Laboratory for constructive discussions.


   Footnotes
 
Supported by Schepens Eye Research Institute Ocular Gene Therapy Program.

Submitted for publication May 25, 2001; revised September 18, 2001; accepted October 18, 2001.

Commercial relationships policy: N.

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: Andrius Kazlauskas, The Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114; kazlauskas{at}vision.eri.harvard.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Committee, RST (1983) The classification of retinal detachment with proliferative vitreoretinopathy Ophthalmology 90,121-125[Medline][Order article via Infotrieve]
  2. Michels, RG, Wilkinson, CP, Rice, TA (1990) Proliferative Retinopathy. Retinal Detachment ,669-706 Mosby St. Louis.
  3. Pastor, JC (1998) Proliferative vitreoretinopathy: an overview Surv Ophthalmol 43,3-18[Medline][Order article via Infotrieve]
  4. Andrews, A, Balciunaite, E, Leong, FL, et al (1999) Platelet-derived growth factor plays a key role in proliferative vitreoretinopathy Invest Ophthalmol Vis Sci 40,2683-2689[Abstract/Free Full Text]
  5. Baudouin, C, Fredj-Reygrobellet, D, Gordon, WC, et al (1990) Immunohistologic study of epiretinal membranes in proliferative vitreoretinopathy Am J Ophthalmol 110,593-598[Medline][Order article via Infotrieve]
  6. Briggs, MC, Grierson, I, Hiscott, P, Hunt, JA (2000) Active scatter factor (HGF/SF) in proliferative vitreoretinal disease Invest Ophthalmol Vis Sci 41,3085-3094[Abstract/Free Full Text]
  7. Cassidy, L, Barry, P, Shaw, C, Duffy, J, Kennedy, S. (1998) Platelet derived growth factor and fibroblast growth factor basic levels in the vitreous of patients with vitreoretinal disorders Br J Ophthalmol 82,181-185[Abstract/Free Full Text]
  8. Connor, TB, Roberts, AB, Sporn, MB, et al (1989) Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye J Clin Invest 83,1661-1666
  9. Garcia-Layana, A, Pastor, JC, Saornil, MA, Gonzalez, G. (1997) Porcine model of proliferative vitreoretinopathy with platelets Curr Eye Res 16,556-563[Medline][Order article via Infotrieve]
  10. Ikuno, Y, Leong, FL, Kazlauskas, A. (2000) Attenuation of experimental proliferative vitreoretinopathy by inhibiting the platelet-derived growth factor receptor Invest Ophthalmol Vis Sci 41,3107-3116[Abstract/Free Full Text]
  11. Kon, CH, Occleston, NL, Aylward, GW, Khaw, PT (1999) Expression of vitreous cytokines in proliferative vitreoretinopathy: a prospective study Invest Ophthalmol Vis Sci 40,705-712[Abstract]
  12. Lashkari, K, Rahimi, N, Kazlauskas, A. (1999) Hepatocyte growth factor receptor in human RPE cells: implications in proliferative vitreoretinopathy Invest Ophthalmol Vis Sci 40,149-156[Abstract/Free Full Text]
  13. Limb, GA, Little, BC, Meager, A, et al (1991) Cytokines in proliferative vitreoretinopathy Eye 5,686-693
  14. Robbins, SG, Mixon, RN, Wilson, DJ, et al (1994) Platelet-derived growth factor ligands and receptors immunolocalized in proliferative retinal diseases Invest Ophthalmol Vis Sci 35,3649-3663[Abstract/Free Full Text]
  15. Claesson-Welsh, L. (1994) Platelet-derived growth factor receptor signals J Biol Chem 269,32023-32026[Free Full Text]
  16. Kazlauskas, A. (2000) A new member of an old family (news; comment) Nat Cell Biol 2,E78-E79[Medline][Order article via Infotrieve]
  17. Li, X, Ponten, A, Aase, K, et al (2000) PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor (see comments) Nat Cell Biol 2,302-309[Medline][Order article via Infotrieve]
  18. Rosenkranz, S, Kazlauskas, A. (1999) Evidence for distinct signaling properties and biological responses induced by the PDGF receptor alpha and beta subtypes Growth Factors 16,201-216[Medline][Order article via Infotrieve]
  19. Rosenkranz, S, DeMali, KA, Gelderloos, JA, Bazenet, C, Kazlauskas, A. (1999) Identification of the receptor-associated signaling enzymes that are required for platelet-derived growth factor-AA-dependent chemotaxis and DNA synthesis J Biol Chem 274,28335-28343[Abstract/Free Full Text]
  20. Rhee, SG, Suh, P-G, Ruy, S-H, Lee, SY (1989) Studies of inositol phospholipid-specific phospholipase C Science 244,546-550[Abstract/Free Full Text]
  21. Rameh, LE, Cantley, LC (1999) The role of phosphoinositide 3-kinase lipid products in cell function J Biol Chem 274,8347-8350[Free Full Text]
  22. Valius, M, Kazlauskas, A. (1993) Phospholipase C-{gamma}1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor’s mitogenic signal Cell 73,321-334[Medline][Order article via Infotrieve]
  23. Wennstrom, S, Siegbahn, A, Yokote, K, et al (1994) Membrane ruffling and chemotaxis transduced by the PDGF beta-receptor require the binding site for phosphatidylinositol 3' kinase Oncogene 9,651-660[Medline][Order article via Infotrieve]
  24. Yao, R, Cooper, GM (1995) Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor Science 267,2003-2006[Abstract/Free Full Text]
  25. Heuchel, R, Berg, A, Tallquist, M, et al (1999) Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3' kinase signaling Proc Natl Acad Sci USA 96,11410-11415[Abstract/Free Full Text]
  26. Tallquist, MD, Klinghoffer, RA, Heuchel, R, et al (2000) Retention of PDGFR-beta function in mice in the absence of phosphatidylinositol 3'-kinase and phospholipase Cgamma signaling pathways Genes Dev 14,3179-3190[Abstract/Free Full Text]
  27. Jerdan, JA, Pepose, JS, Michels, RG, et al (1989) Proliferative vitreoretinopathy membranes Ophthalmology 9,801-810
  28. Schiro, JA, Chan, BM, Roswit, WT, et al (1991) Integrin alpha 2 beta 1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells Cell 67,403-410[Medline][Order article via Infotrieve]
  29. Yang, CH, Huang, TF, Liu, KR, Chen, MS, Hung, PT (1996) Inhibition of cell-induced vitreous contraction by synthetic peptide derived from the collagen receptor binding sequence J Ocul Pharmacol Ther 12,353-361[Medline][Order article via Infotrieve]
  30. Yang, CH, Huang, TF, Liu, KR, Chen, MS, Hung, PT (1996) Inhibition of retinal pigment epithelial cell-induced tractional retinal detachment by disintegrins, a group of Arg-Gly-Asp-containing peptides from viper venom Invest Ophthalmol Vis Sci 37,843-854[Abstract/Free Full Text]
  31. Yang, CH, Liu, CZ, Huang, TF, et al (1997) Inhibition of RPE cell-mediated matrix adhesion and collagen gel contraction by crovidisin, a collagen-binding snake venom protein Curr Eye Res 16,1119-1126[Medline][Order article via Infotrieve]
  32. Gelderloos, JA, Rosenkranz, S, Bazenet, C, Kazlauskas, A. (1998) A role for Src in signal relay by the platelet-derived growth factor alpha receptor J Biol Chem 273,5908-5915[Abstract/Free Full Text]
  33. Ory, DS, Neugeboren, BA, Mulligan, RC (1996) A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes Proc Natl Acad Sci USA 93,11400-11406[Abstract/Free Full Text]
  34. Bazenet, CE, Gelderloos, JA, Kazlauskas, A. (1996) Phosphorylation of tyrosine 720 in the platelet-derived growth factor alpha receptor is required for binding of Grb2 and SHP-2 but not for activation of Ras or cell proliferation Mol Cell Biol 16,6926-6936[Abstract]
  35. Bazenet, C, Kazlauskas, A. (1994) The PDGF receptor alpha subunit activates p21ras and triggers DNA synthesis without interacting with rasGAP Oncogene 9,517-525[Medline][Order article via Infotrieve]
  36. Valius, M, Bazenet, C, Kazlauskas, A. (1993) Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor ß subunit and are required for binding of phospholipase C{gamma} and a 64-kilodalton protein, respectively Mol Cell Biol 13,133-143[Abstract/Free Full Text]
  37. Kazlauskas, A, Cooper, JA (1990) Phosphorylation of the PDGF receptor ß subunit creates a tight binding site for phosphatidylinositol 3 kinase EMBO J 9,3279-3266[Medline][Order article via Infotrieve]
  38. Skuta, G, Ho, CH, Grinnell, F. (1999) Increased myosin light chain phosphorylation is not required for growth factor stimulation of collagen matrix contraction J Biol Chem 274,30163-30168[Abstract/Free Full Text]
  39. Nakagawa, M, Refojo, MF, Marin, JF, Doi, M, Tolentino, FI (1995) Retinoic acid in silicone and silicone-fluorosilicone copolymer oils in a rabbit model of proliferative vitreoretinopathy Invest Ophthalmol Vis Sci 36,2388-2395[Abstract/Free Full Text]
  40. Fastenberg, DM, Diddie, KR, Sorgente, N, Ryan, SJ (1982) A comparison of different cellular inocula in an experimental model of massive periretinal proliferation Am J Ophthalmol 93,559-564[Medline][Order article via Infotrieve]
  41. Hida, T, Chandler, DB, Sheta, SM (1987) Classification of the stages of proliferative vitreoretinopathy in a refined experimental model in the rabbit eye Graefes Arch Clin Exp Ophthalmol 225,303-307[Medline][Order article via Infotrieve]
  42. Rosenkranz, S, Ikuno, Y, Leong, FL, et al (2000) Src family kinases negatively regulate platelet-derived growth factor alpha receptor-dependent signaling and disease progression J Biol Chem 275,9620-9627[Abstract/Free Full Text]
  43. Ahlen, K, Berg, A, Stiger, F, et al (1998) Cell interactions with collagen matrices in vivo and in vitro depend on phosphatidylinositol 3-kinase and free cytoplasmic calcium Cell Adhes Commun 5,461-473[Medline][Order article via Infotrieve]
  44. Campochiaro, PA (1997) Mechanisms in ophthalmic disease: pathogenic mechanisms in proliferative vitreoretinopathy Arch Ophthalmol 115,237-241[Abstract]
  45. Ryan, SJ (1993) Traction retinal detachment: XLIX Edward Jackson Memorial Lecture Am J Ophthalmol 115,1-20[Medline][Order article via Infotrieve]
  46. Soriano, P. (1994) Abnormal kidney development and hematological disorders in PDGF ß-receptor mutant mice Genes Dev 8,1888-1896[Abstract/Free Full Text]
  47. Soriano, P. (1997) The PDGF alpha receptor is required for neural crest cell development and for normal patterning of the somites Development 124,2691-2700[Abstract]
  48. Casaroli, Marano RP, Vilaro, S (1994) The role of fibronectin, laminin, vitronectin and their receptors on cellular adhesion in proliferative vitreoretinopathy Invest Ophthalmol Vis Sci 35,2791-2803[Abstract/Free Full Text]
  49. Chrzanowska-Wodnicka, M, Burridge, K. (1996) Rho-stimulated contractility drives the formation of stress fibers and focal adhesions J Cell Biol 133,1403-1415[Abstract/Free Full Text]
  50. Goeckeler, ZM, Wysolmerski, RB (1995) Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation J Cell Biol 130,613-627[Abstract/Free Full Text]
  51. Kreisberg, JI, Venkatachalam, MA (1986) Vasoactive agents affect mesangial cell adhesion Am J Physiol 251,C505-C511[Abstract/Free Full Text]
  52. Lamb, NJ, Fernandez, A, Conti, MA, et al (1988) Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase J Cell Biol 106,1955-1971[Abstract/Free Full Text]



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