of Medicine, Baltimore, Maryland. Supported by The Core ... 600 N Wolfe Street,. Baltimore, MD 21287. ..... Ashton N. Neovascularization in ocular disease.Trans.
Activation of Protein Tyrosine Phosphorylation After Retinal Branch Vein Occlusion in Cats Atsushi Hayashi, Kazuyuki Imai, Hyung Chan Kim, and Eugene dejuan, Jr.
Purpose. The authors examine the effect of retinal branch vein occlusion (BVO), a common retinal vascular disorder, on protein tyrosine phosphorylation, production of angiogenic growth factors, and activation of signal proteins in the tyrosine kinase pathways in the retina. Methods. Retinal branch vein occlusion was induced in cat retina by coagulation of retinal veins with diathermy. At 2 days, 1, 3, and 6 weeks after induction of BVO, the retina was divided into three parts: a part within the distribution of the occluded vein (BVO[IN]) or a part outside the distribution of the occluded vein (BVO [OUT]). Each part of the retina was prepared for Western blot analysis of tyrosine-phosphorylated proteins, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and four signal proteins in the tyrosine kinase pathways, which were phospholipase Cy (PLCy), C-Src, SH2-containing protein (SHC), and mitogen-activated protein kinase (MAPK). Results. Overall, tyrosine-phosphorylated proteins were increased after BVO, especially in BVO (IN) at 2 days and 1 week. The VEGF and bFGF also were increased in BVO (IN) at 1 week and 2 days, respectively. The PLCy and MAPK were activated at these time points. The C-Src and SHC were not activated in the retina after BVO. Conclusions. The BVO increased overall protein tyrosine phosphorylation in the cat retina in association with increase of angiogenic growth factors (VEGF and bFGF) and activation of two signal proteins (PLCy and MAPK) in the tyrosine kinase pathways. These results suggest that the protein tyrosine phosphorylation may in part play an important role in mitogenesis of vascular endothelial cells and other retinal responses after BVO. Invest Ophthalmol Vis
Sci. 1997; 38:372-380.
JVetinal branch vein occlusion (BVO) is a frequent, common retinal vascular disease and can be associated with ocular complications such as retinal neovascularization and macular edema, which cause loss of visual acuity.1"4 Retinal neovascularization is observed in approximately 16% to 30% of patients with BVO,1'4 and the macular edema is observed in approximately 50% to 60% of patients with BVO.12 Retinal ischemia is thought to be a major reason for the resultant proliferative responses after retinal vein occlusion, including
From the Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Supported by The Core Grant of the Johns Hopkins University (P01-EY01765) and The Man Poiuer Award of Research to Prevent Blindness. Submitted for publication June 13, 1996; revised August 23, 1996; accepted Se/Hember 11, 1996. Proprietary interest category: N. Reprint requests: Eugene dejuan, Jr., Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine, Maumenee 738, 600 N Wolfe Street, Baltimore, MD 21287.
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retinal neovascularization,5 7 and capillary nonperfusion has been shown to correlate with the incidence of retinal neovascularization.4 Experimental studies of BVO, using monkeys and cats, have reported the histopathologic characteristics in the retina after BVO.8"11 Within the distribution of the occluded retinal vein in the monkey retina, there was an increase in the density of vascular endothelial cells but a decrease in the density of pericytes.12 We previously have shown that proliferation of endothelial cells in retinal venules and capillaries increased within the distribution of the occluded vein and that retinal homogenates of this affected area had mitogenic activity to endothelial cells in vitro.13'14 However, it is not fully understood what causes proliferation of endothelial cells in the retina after retinal vein occlusion or which signal transduction pathways are activated. Peptide growth factors have been shown to induce
Investigative Ophthalmology & Visual Science, February 1997, Vol. 38, No. 2 Copyright © Association for Research in Vision and Ophthalmology
Phosphotyrosine Induction After Branch Vein Occlusion in Cats proliferation of retinal capillary endothelial cells in vitro and in vivo.13""'5 Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have been shown to be potent mitogens for vascular endothelial cells15'16 and are produced in association with retinal ischemia.17"19 Recently, VEGF levels were shown to correlate with retinal ischemia and iris neovascularization.19'20 Most angiogenic growth factors, including VEGF and bFGF, are known to bind specific cell surface receptors with intrinsic tyrosine kinases, which in turn cause the activation of Src homology 2 (SH2) and Src homology 3 (SH3) domain containing proteins in the tyrosine kinase pathways and induce cell proliferation and other cellular responses.21"23 We have shown that tyrosine phosphorylation of retinal capillary endothelial cells plays an important role in their proliferation.24'25 Several signal proteins in the tyrosine kinase pathways such as phospholipase Cy (PLCy), SH2-containing protein (SHC), and C-Src have been shown to mediate mitogenic signal transductions through activating Ras signaling pathways and protein kinases such as protein kinase C and S6 kinase.21"23'26 Mitogen-activated protein kinase (MAPK) also is an important protein kinase in signaling pathways downstream from Ras protein and induces cell proliferation or differentiation by phosphorylating a number of transcription factors and protein kinases.27"29 In the present study, to clarify the biochemical events induced by BVO, we used the established BVO model in cats and examined whether protein tyrosine phosphorylation is induced by BVO, whether BVO increases angiogenic growth factors such as VEGF and bFGF, and which signal proteins in the tyrosine kinase pathways are activated by BVO (PLCy, C-Src, SHC, and MAPK). METHODS Induction of Retinal Branch Vein Occlusion Eight adult male cats (weighing 2 to 3 kg) were used for induction of retinal BVO as methods described previously."'13'14 Cats were anesthetized with ketamine (40 mg/kg) and acepromazine (1.6 mg/kg). The pupil was dilated with 1% tropicamide and 10% phenylephrine. Under an operating microscope, a small incision of the conjunctiva in one eye was made and the sclera at the pars plana was perforated with a 20-gauge needle into the vitreous cavity. With the aid of a contact lens on the cornea, a bipolar coaxial diathermy probe was introduced into the vitreous and one or two major retinal veins were coagulated about at 1 disc diameter apart from the optic disc. The current was adjusted to the minimum level to produce con-
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striction of retinal veins. The BVO was induced on upper and temporal or nasal veins. Just after occlusion of retinal veins, the veins showed enlargement and tortuosity in the peripheral portion from the coagulated site. After coagulation, the probe was taken out from the eye and one 8-0 silk suture was placed to close the incision. Gentamicin (5 mg) was injected into the subconjunctival space, and an antibiotic ointment was put on the cornea and the conjunctiva. After the surgery, the cat eyes were observed periodically with an indirect ophthalmoscope. Cats were killed at 2 days, 1, 3, and 6 weeks after induction of BVO. A fluorescein angiogram was taken with cats at 6 weeks after BVO as described previously.1314 Normal eyes that were not operated on served as control specimens. No sham operations were performed. All procedures conformed to the Association for Research in Vision and Ophthalmolog)' statements for the Use of Animals in Ophthalmic and Vision Research. Sample Preparation Normal eyes and the eyes after 2 days, 1, 3, and 6 weeks after induction of BVO were enucleated, and the anterior segments and the vitreous were removed. Each retina was divided into three parts (approximately one third of the retina) so that each part of the retina included either occluded or nonoccluded major retinal vein. The retina was dissected carefully from the pigmented epithelium and the choroid. If a retinal area contained pigment, it was not included in the sample preparation. The procedures for sample preparation were described previously.24'25'30 In brief, the isolated retina was immersed in ice-cold lysis buffer (150-mM NaCl, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50-mM Tris, 100 fig/m\ phenylmethylsulfonyl fluoride, 0.3 //g/ml ethylenediaminetetraacetic acid, 0.7 (J,g/m\ pepstatin A, 0.5 //g/ml leupeptin, 1-mM orthovanadate, and 50-//M sodium fluoride) and homogenized. An equal amount of 2X SDS sample buffer was added to the lysate. The 2X SDS sample buffer was made up of 160-mM Tris (pH 6.8), 4% SDS, 30% glycerol, 5% /?-mercaptoethanol, 10-mM dithiothreitol, and 0.01% bromophenol blue. The samples were boiled at 95°C for 5 minutes and centrifuged at 13,000 rpm for 10 minutes. The supernatants were collected and stored at — 80°C. After protein concentrations of these total retinal samples were measured by the Pierce BCA method, they were used for gel electrophoresis and Western blot analysis. Antibodies Used for Analysis The following primary antibodies were used for Western blot analysis: mouse monoclonal antiphosphotyro-
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tion and rinsed with the lysis buffer three times. The pellets were resuspended in 150 //I of IX SDS sample buffer per 1 mg of retinal homogenate and boiled at 95°C for 5 minutes. The supernatants were collected and stored at —80°C. These immunoprecipitated samples were used for gel electrophoresis and Western blot analysis to examine tyrosine phosphorylation of intracellular signal proteins in the tyrosine kinase pathways. Gel Electrophoresis
FIGURE l. A fluorescein angiogram of a cat retina at 6 weeks after induction of retinal branch vein occlusion. The coagulated site of the retinal vein was occluded completely {mroioheads). Shunt vessel formation and capillary nonperfusion areas in the retina within the distribution of the occluded vein were indicated by arrows and stars, respectively.
sine antibody (PY20) (1:500 dilution) (Transduction laboratories, Lexington, KY); rabbit antihuman vascular endothelial growth factor antibody (1:100 dilution), rabbit antihuman C-Src antibody (1:100 dilution), and rabbit antibovine phospholipase Cyl antibody (1:100 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit antirat mitogen-activated protein kinase antibody R2 (1:2000 dilution), and rabbit antihuman SHC antibody (1:1000 dilution) (Upstate Biotechnology, Lake Placid, NY). We also used a rabbit antibovine basic fibroblast growth factor antibody (1:2000 dilution) (Sigma Chemical Company, St. Louis, MO). Immunoprecipitation Another four cats, in which BVO was induced as described above, were killed at 2 days, 1, 3, and 6 weeks after induction of BVO. The procedures of immunoprecipitation were described previously."4'25'30 Briefly, each part of the retina was collected in an ice-cold lysis buffer (10-mM Tris pH 7.4, 1-mM ethylenediaminetetraacetic acid, 1-mM EGTA, 150-rnM NaCl, 1% Triton-X 100, 0.5% NP-40, 100 /xg/ml phenylmethylsulfonyl fluoride) and homogenized. Protein concentrations were measured by the Pierce BCA method. Then, 20 fj,g of agarose conjugated antiphosphotyrosine antibody (PY20) (Santa Cruz Biotechnology) was added to each 1 mg of retinal homogenate. Retinal homogenates were incubated with the agarose-conjugated antiphosphotyrosine antibody at 4°C overnight with shaking. The pellets were collected by centrifuga-
The procedures of gel electrophoresis were described previously.24'2030 In brief, equal protein amount in each sample was electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The samples were run on 4% to 20% gradient mini gels (BioRad Laboratories, Hercules, CA) with the use of a BIORAD Protean II apparatus. Biotinylated and prestained molecular markers (Bio-Rad) were run with the samples simultaneously. After electrophoresis, gels were processed either for total protein staining with Coomassie brilliant blue dye or for Western blot analysis. Coomassie Brilliant Blue Staining After gel electrophoresis, the gels were fixed in 45% methanol and 10% acetic acid aqueous solution for 30 minutes. Then they were soaked in saturated picric acid solution briefly and stained with 0.25% aqueous Coomassie brilliant blue (R-250) solution for a minimum of 2 hours. The gels were destained in 10% acetic acid solution. The procedure was repeated three times. Western Blot Procedures For Western blot analysis, the gels were electroblotted onto nitrocellulose membranes (Costar Scientific, Cambridge, MA) with the use of a transblot semi-dry apparatus (Bio-Rad). The membranes for detection of tyrosine-phosphorylated proteins were incubated with 3% bovine serum albumin in Tris-buffered saline (TBS) (20-mM Tris and 150-mM NaCl, pH 7.5) for 1 hour at room temperature. The membranes for detection of other proteins were incubated with 2% of nonfat dried milk (Bio-Rad) in TBS for 1 hour at room temperature. Then each membrane was incubated with a primary antibody solution at 4°C overnight. After membranes were rinsed three times with TBS, they were incubated with a solution of horseradish peroxidase-conjugated goat antimouse immunoglobulin G antibody or antirabbit immunoglobulin G antibody (Bio-Rad) (1:2000 dilution) and avidin-horseradish peroxidase (Bio-Rad) (1:15000 dilution) for 2 hours at room temperature. Avidin-horseradish peroxidase was used for detection of biotinylated molecular
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Changes of Total Protein Profiles After Branch Vein Occlusion After measurement of protein concentrations, equal protein amounts of the retinal samples of BVO (IN) and a part outside the distribution of the occluded vein [BVO(OUT)] at 2 days, 1, 3, and 6 weeks after BVO were loaded in the same gel. Coomassie brilliant blue staining of the gel showed changes of total protein profiles in BVO (IN) and BVO (OUT) (Fig. 2), In total protein profiles, at least two bands increased after BVO. These two bands had approximate molecular weights of 72 kDa and 14 kDa. The 72-kDa band in BVO (IN) at 2 days and 1 week was stronger than that in BVO (OUT), and a marked increase of the 72-kDa band was detected in BVO (IN) at 2 days. After 3 weeks, the 72-kDa band was decreased and showed no difference between BVO(IN) and BVO(OUT). The I4-kDa band was prominent in BVO (IN) at 2 days and 1 and 3 weeks and was stronger in BVO (IN) than in BVO (OUT).
FIGURE 2. Protein profiles of retinas from normal cat eyes and eyes obtained at 2 days and 1, 3, and 6 weeks (indicated by the number at the top of each lane) after retinal branch vein occlusion. The retinas from each group of cat eyes were processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gel was stained with Coomassie brilliant blue. Molecular weights (in kilodaltons) are shown to the left. I = the retinal area within the distribution of a occluded vein; O = the retinal area outside the distribution Increase of Tyrosine-Phosphorylated Proteins of a occluded vein; C = control. Two bands of 72 kDa and After Branch Vein Occlusion 14 kDa {arrowheads) were increased after induction of branch vein occlusion. The samples then were examined by Western blot
markers. Finally, after being rinsed with TBS containing 0.3% Tween-20 three times, followed by rinsing with TBS twice, the membranes were reacted with enhanced chemiluminescence reagents (Amersham Life Science, Arlington Heights, IL) and exposed to Kodak x-ray films (Eastman Kodak, Rochester, NY) for 15 seconds to 5 minutes, as described in Amersham enhanced chemiluminescence protocols. Each Western blot analysis was repeated at least three times.
analysis with antiphosphotyrosine antibody (PY20) (Fig. 3). As shown in Figure 3, three major immunoreactive tyrosine-phosphorylated bands were detected at approximate molecular weights of 140 kDa, 110 kDa, and 63 kDa. The 110-kDa band was increased in BVO (IN) at 2 days after BVO compared to that in 2d C
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116-64 M RESULTS Clinical Appearance of Branch Vein Occlusion The immediate venous engorgement and tortuosity developed in all eyes after induction of BVO. Extensive retinal hemorrhage, retinal edema, and exudative retinal detachment were observed in the retina within the distribution of the occluded vein [BVO(IN)] by 2 days. The exudative retinal detachment absorbed spontaneously 2 to 3 weeks after the induction of the BVO. The retinal hemorrhage also absorbed by 6 weeks after BVO. Figure 1 shows a fluorescein angiogram of a cat retina at 6 weeks after induction of BVO. The complete occlusion is shown at the site of coagulation on the retinal vein. Formation of shunt vessels, capillary nonperfusion, and capillary dilatation also was observed in BVO (IN).
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lecular weights were 28 kDa (p28), 26 kDa (p26), and 14 kDa (pl4). The p28 and p26 were detected in all retinal samples. However, the pl4 was increased strongly in BVO (IN) at 2 days.
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FIGURE 4. Western blots of vascular endothelial growth factor (A) and basic fibroblast growth factor (B) in samples of cat retinas after retinal branch vein occlusion. Molecular weights of the proteins are shown to the left (in kilodaltons), and time after branch vein occlusion is shown at the top of each blot. C = control; I = the retinal area within the distribution of a occluded vein; O = the retinal area outside the distribution of a occluded vein. (A) Western blot of vascular endothelial growth factor after branch vein occlusion. Two forms of vascular endothelial growth factor were detected at 29 kDa (p29) and 15 kDa (pi5) of molecular weights (arrows). (B) Western blot of basic fibroblast growth factor after branch vein occlusion. Three forms of basic fibroblast growth factor were detected at 28 kDa (p28), 26 kDa (p26), and 14 kDa (pl4) of molecular weights {arrows).
the control specimen and in BVO (OUT). The 63-kDa band was increased in BVO (IN) and BVO (OUT) at 2 days and 1 week after BVO compared to that in the control specimen. The tyrosine-phosphorylated bands at 6 weeks after BVO were almost similar to those in die control specimen. Increase of Angiogenic Growth Factors After Branch Vein Occlusion Vascular Endothelial Growth Factor. Figure 4A shows a Western blot analysis of vascular endothelial growth factor (VEGF) in the retinas after BVO. Two major bands of VEGF were detected, and their approximate molecular weights were 29 kDa (p29) and 15 kDa (pl5). Although the p29 was detected in all retinal samples, the pi5 was increased strongly in BVO (IN) at 1 week. Basic Fibroblast Growth Factor. Figure 4B shows a Western blot analysis of basic fibroblast growth factor (bFGF) in the retinas after BVO. Three bands of bFGF were detected in the retina. Those approximate mo-
Changes of Signal Proteins in Tyrosine Kinase Pathways After Branch Vein Occlusion Phospholipase Cy. Figure 5A shows Western blot analyses of total protein changes of phospholipase Cy (PLCy) (T) and of its tyrosine-phosphorylated form (IP). The approximate molecular weight of PLCy was 145 kDa (pl45). The total protein amount of PLCy (T) was increased after the BVO induction, especially in BVO (IN) at 3 and 6 weeks after BVO. The tyrosinephosphorylated form of PLCy (IP) also was increased in BVO (IN) and BVO (OUT) at 2 days and 1 week after BVO. After 3 weeks of BVO, it was decreased and similar to that of the control specimen. C-Src. Figure 5B shows Western blot analyses of total protein changes of C-Src (T) and of its tyrosinephosphorylated form (IP). The C-Src had an approximate molecular weight of 60 kDa (p60). The total protein amount of C-Src (T) (p60) appeared increased as evident by stronger bands of p60 in BVO (OUT) than in BVO (IN), although the p60 band was stronger in BVO (IN) than in BVO (OUT) at 3 weeks after BVO. The tyrosine-phosphorylated form of p60 (IP) in BVO (OUT) also showed a similar tendency, but the tyrosine-phosphorylated p60 band was the same in BVO (IN) and BVO (OUT) by 6 weeks after BVO. SH2-Containing Protein. Figure 5C shows Western blot analyses of total protein changes of SHC (T) and of its tyrosine-phosphorylated form (IP). As shown in Figure 5C, three bands of SHC were detected in total protein samples (T) at approximate molecular weights of 66 kDa (p66), 52 kDa (p52), and 46 kDa (p46). The total protein amounts of SHC in BVO (IN) were increased, compared to those in BVO (OUT) at corresponding time points, but the apparent amounts of SHC were not different at 6 weeks after BVO. The tyrosine-phosphorylated form of SHC (p52) was not changed after BVO (IP). The p52 band of SHC was the major tyrosine-phosphorylated form in cat retina. Mitogen-Activated Protein Kinase. Figure 5D shows Western blot analyses of total protein changes of MAPK (T) and of its tyrosine-phosphorylated form (IP). The total protein amounts of MAPK were increased after BVO, especially in BVO (IN) after 3 weeks. The tyrosine-phosphorylated forms of MAPK (IP) had approximate molecular weights of 44 kDa (p44) and 42 kDa (p42). The tyrosine-phosphorylated form of p44 increased after BVO, especially in BVO (IN) and BVO (OUT) at 2 days and 1 week. The tyrosine-phosphorylated form of p42 only was de-
Phosphotyrosine Induction After Branch Vein Occlusion in Cats
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tected in BVO (IN) and BVO (OUT) at 2 days and 1 week. Another band was detected in the immunoprecipitated samples (IP), but this was regarded as an unidentified MAPK-like protein or a nonspecific band because of its high molecular weight. DISCUSSION In the present study, we used a retinal BVO model in cats because this model has been examined histopathologically and its clinical and histologic observations are similar to those of BVO in human disease.1011'13 We and others have observed similar retinal changes in human BVO, such as engorgement and tortuosity of occluded
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FIGURE 5. Western blots of four signal proteins in the tyrosine kinase pathways after retinal branch vein occlusion (BVO). Molecular weights of the proteins are shown to the left (in kilodaltons), and time after BVO is shown at the top of each blot. C = control; I = the retinal area within the distribution of a occluded vein; O = the retinal area outside the distribution of a occluded vein. Western blots of total retina] samples are shown as T, and Western blots of samples immunoprecipitated with antiphosphotyrosine antibody (PY20) are shown as tyrosine-phosphorylated form (IP). (A) Western blots of phospholipase Cy (PLCy) after BVO. PLCy was detected at 145 kDa (pl45) of molecular weight {arrow). (B) Western blots of C-Src after BVO. C-Src was detected at 60 kDa (p60) of molecular weight {arrow). (C) Western blots of SH2-containing protein after BVO. Three forms of SH2-containing protein were detected in T at 66 kDa (p66), 52 kDa (p52), and 46 kDa (p46) of molecular weights (arrows). One form of SH2-containing protein (52 kDa) was detected in I (arrow). (D) Western blots of mi togen-activated protein kinase after BVO. One mitogen-activated protein kinase band was detected in T at 44 kDa (p44) of molecular weight (arrow). Two forms of mitogen-activated protein kinase were detected in I at 44 kDa and 42 kDa (arrows). A mitogen-activated protein kinase-like protein or nonspecific band is shown (arrowhead).
retinal veins, retinal hemorrhage, retinal edema, and exudative retinal detachment in the retina within the distribution of occluded veins [BVO(IN)].1-33132 Fluorescein angiography also showed changes similar to those of human BVO, such as capillary nonperfusion, formation of shunt vessels, and staining of occluded veins in BVO(IN). Although we did not observe retinal or iris neovascularization after induction of BVO in cats, this model provides a useful experimental tool to examine retinal changes associated with BVO. In the total protein profiles in the retinas after BVO, we found two bands (72 and 14 kDa) that were increased, especially in BVO (IN). These bands have not been specified yet, but we speculate that these bands may represent ischemia-induced proteins, because we detected a similar increase of the 72-kDa band in the total protein profiles of rat retinas after ischemia-reperfusion injury.30 However, further investigation is needed to specify these proteins. We showed that injury of retinal vein occlusion causes an increase of protein tyrosine phosphorylation in the retina. Tyrosine-phosphorylated proteins were increased, especially in BVO (IN), at approximate molecular weights of 110 and 63 kDa. Interestingly, the 63-kDa band showed the same molecular weight that we detected in the Western blot analysis of tyrosinephosphorylated proteins in the rat retina after ischemia-reperfusion injury.30 Although it is unknown what kinds of proteins are included in these bands,
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the retinal ischemia may cause a similar increase of tyrosine-phosphorylated proteins, even though the ways to induce retinal ischemia are different. We also have shown an increase in tyrosine-phosphorylated proteins in the retina in the BVO(OUT) at 1 week compared to that of the control retina. This increase in tyrosine-phosphorylated proteins in BVO(OUT) partly may be because of an exudative retinal detachment that extended from BVO(IN). Our results suggest that both retinal degeneration and retinal ischemia caused by retinal vein occlusion may participate in the increase in protein tyrosine phosphorylation of the retina. Because the relative amount of tyrosinephosphorylated proteins is larger in BVO(IN) than in BVO(OUT) (Fig. 3), we regarded the retinal ischemia (represented by capillary nonperfusion) to be a major cause of the increase in protein tyrosine phosphorylation of the retina after BVO. To examine a relation between the increase in protein tyrosine phosphorylation after BVO and the proliferation of vascular endothelial cells, we studied the changes in levels of two angiogenic growth factors, VEGF and bFGF, in the retina after BVO. We chose these two growth factors because VEGF and bFGF are potent mitogens for vascular endothelial cells,15 their mRNA expressions have been shown to be in part regulated by retinal ischemia,161719'33 and they bind to specific cell surface receptors with intrinsic tyrosine kinases and activate the tyrosine kinase pathways.21"23 We found a marked increase of the 15 kDa of VEGF (pl5) in BVO (IN) at 1 week alter BVO. This pl5 has been suggested to be a secreted form of VEGF34 or derived from proteolysis of higher molecular-weight forms of VEGF.16'35 We have not clarified whether this pl5 was derived from secretion or proteolysis, but the decrease of the 29 kDa of VEGF in BVO (IN) at the same time point suggests that this pl5 might be derived from in vivo proteolysis. Because this increase was not detected in BVO (OUT) at 1 week after BVO, the increase of pi 5 may be caused by retinal vein occlusion. We also found an increase of the 14-kDa band of bFGF (pi 4) in BVO (IN) at 2 days, which corresponded to the previous studies.36'37 Other studies have detected low molecular-weight forms of bFGF, such as pi 4 in rat brain36'37 and in hepatoma cells.38 The low molecular-weight forms of bFGF were suggested to be produced by protease in the nucleus,37'39 but they have been shown to have die same mitogenic activity as that of the 18 kDa of bFGF.38 Because we used a variety of protease inhibitors throughout the procedures of sample preparation, these pl5 of VEGF and pl4 of bFGF may be derived in vivo by protease in the retina. Because we showed an increase of protein tyrosine phosphorylation after BVO, we examined whether
four important signal proteins in the tyrosine kinase pathways are activated in the retina after BVO.21"23'26 We showed an increase of tyrosine-phosphorylated forms of two signal proteins, which were PLCy and MAPK, in BVO (IN) and BVO (OUT) at 2 days and 1 week after BVO. Because PLCy has been shown to be activated by bFGF, VEGF, and other growth factors and mediate mitogenic signals through activation of protein kinase C,22'26 the increase of tyrosine-phosphorylated form of PLCy may support the increase in mitogenesis of retinal cells, such as vascular endothelial cells described previously.1213 MAPK also has been shown to play crucial roles in cell proliferation and differentiation.27"29 The increase of tyrosine-phosphorylated form of MAPK also may support increased mitogenesis in the retina after BVO.13 Protein kinase C has been shown to participate in activation of MAPK through Ras signaling pathways.22'28 Therefore, activation of PLCy may contribute to further activation of MAPK. Interestingly, we also showed an increase of tyrosine-phosphorylated proteins in BVO (OUT) at 1 week. Consistent with this result, the tyrosine-phosphorylated forms of PLCy and MAPK were increased in BVO (OUT). Recent studies have shown that the expression of VEGF was induced in the detached retina and that the expression of platelet-derived growth factor was induced in the retinal pigment epithelium underlying retinal detachment.18'40 Our results suggest that the increase in tyrosine phosphorylation of PLCy and MAPK can be induced by both the retinal ischemia with retinal vein occlusion and the exudative retinal detachment. Further studies are needed to clarify the mechanisms of activation of these signal proteins and the correlation between growth factor receptors and intracellular signal proteins. In summary, we showed that BVO increased protein tyrosine phosphorylation, the amount of angiogenic growth factors (VEGF and bFGF), and tyrosine phosphorylation of at least two signal proteins in the tyrosine kinase pathways (PLCy and MAPK) in the retina. These results suggest that these angiogenic growth factors may play a role in retinal responses after retinal vein occlusion, such as proliferation of vascular endothelial cells through protein tyrosine phosphorylation. These results also raise a possibility to prevent pathologic changes induced by BVO by inhibiting excessive protein tyrosine phosphorylation or activation of signal proteins. Key Words cat, growth factors, protein tyrosine phosphorylation, retina, retinal vein occlusion Acknowledgment The authors thank Mr. David E. Baranano for his enthusiasm and his technical assistance in this study.
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