Prostaglandin E2 Induces Vascular Endothelial

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Prostaglandin E2 Induces Vascular Endothelial Growth. Factor and Basic Fibroblast Growth Factor mRNA. Expression in Cultured Rat Muller Cells. Tong Cheng ...

Prostaglandin E2 Induces Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor mRNA Expression in Cultured Rat Muller Cells Tong Cheng,1'2 Wei Cao,1'2 Rong Wen,5 Roy H. Steinberg,1'2 and Matthew M. LaVail2'4 PURPOSE. TO investigate the induction of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene expression by prostaglandin E2 (PGE2) in cultured rat Muller cells and to study the mechanism of the induction. METHODS. Muller cells were obtained from neonatal Sprague-Dawley rat retinas and cultured in essential modified Eagle's medium supplemented with 10% fetal calf serum for up to four passages. Cells were treated with PGE2, protein kinase A (PKA) inhibitors H-89 or SQ 22536, protein kinase C (PKC) inhibitors calphostin C or GF 109203X, PKC activator phorbol 12-myristate 13-acetate (PMA), or the PKA activator forskolin. Northern blot analysis was performed to determine the levels of VEGF and bFGF mRNA. RESULTS. PGE2 induced VEGF and bFGF mRNA expression in a dose- and time-dependent manner. VEGF and bFGF mRNA reached peaks of 2- and 35-fold at 10 /xM PGE2. No further increases were observed at 100 IJM PGE2. When treated with 10 JLIM PGE2, the increases in VEGF and bFGF mRNA reached maximum by 2 hours, then slowly declined toward the control level within 24 hours of PGE2 treatment. The inductions of VEGF and bFGF mRNA expression by PGE2 were blocked by the specific PKA inhibitors H-89 (30 /u,M) or SQ 22536 (500 ^M, 1000 /xM). Forskolin (10 /xM), a cyclic adenosine monophosphate activator, also stimulated VEGF and bFGF mRNA expression. However, the effects of forskolin and PGE2 on VEGF gene expression were not additive, whereas forskolin enhanced the effect of PGE2 on bFGF mRNA expression. The specific PKC inhibitors, GF 109203X (2 jaM) and calphostin C (1 fxM), did not inhibit PGE2-induced VEGF gene expression, whereas PGE2-induced bFGF expression was blocked by the PKC inhibitor GF 109203X. In addition, downregulation of PKC by PMA (0.8 jaM) treatment did not block the induction of VEGF gene expression, whereas it did inhibit the induction of bFGF mRNA expression. CONCLUSIONS. These results indicate that PGE2 stimulates VEGF and bFGF mRNA expression in cultured rat Muller cells. The induction of VEGF seems to occur through activation of the PKA pathway, whereas that of bFGF occurs through PKA and PKC activation. These findings raise the possibility that endogenous PGE2 stimulates VEGF and bFGF mRNA expression in Muller cells in vivo under conditions in which PGE2 production is increased, such as in injury. (Invest Ophthalmol Vis Set. 1998;39:581-591)

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rostaglandins, as pathogenetic mediators, are produced in tissue, including the retina, in response to inflammation at the site of injury. In the acutely injured cat spinal cord or in cultures of cardiac myocytes and of nonmuscle cells, the release of prostaglandins is increased after injury.12 Prostaglandin E2 (PGE2) is the major prostaglandin in the retina.3 Increases in PGE2 were found in various pathologic conditions in the retina, including various retinal injuries, such as laser irradiation,4'5 optic nerve

From the Departments of 'Physiology, 2 Ophthalmology, and ''Anatomy, University of California, San Francisco and the -^Departments of Ophthalmology and Cell and Developmental Biology, University of Pennsylvania, Philadelphia. Supported by National Institutes of Health grants EY01429, EY01919, and EY06842 and by funds from the Foundation Fighting Blindness, Research to Prevent Blindness, and That Man May See. Submitted for publication May 21, 1997; revised November 6, 1997; accepted November 18, 1997. Proprietary interest category: N. Reprint requests: Matthew M. LaVail, Beckman Vision Center, University of California, San Francisco, CA 94143-0730. Investigative Ophthalmology & Visual Science, March 1998, Vol. 39, No. 3 Copyright © Association for Research in Vision and Ophthalmology

injury,6 and retinal detachment.7 Prostaglandins have also been found as mediators in retinal disorders with neovascularization, such as diabetic retinopathy,8 retrolental fibroplasia,9 and retinopathy of prematurity.10 Studies have shown that prostaglandins are capable of inducing angiogenesis.1 '"' 3 In addition to their wellknown role as inflammatory mediator, there is increasing evidence to show that prostaglandins have cytoprotective properties on various tissue types against toxic chemicals."1"17 Cytoprotection of nerve tissue by prostaglandins has also been observed, 18 " 20 but the mechanism mediating this cytoprotection is unknown. Because prostaglandins do not directly stimulate endothelial cell growth, 21 it is speculated that their angiogenic effect may be fulfilled by the paracrine action of angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). On the other hand, because of well-known cytoprotective effects of bFGF, it is possible that die cytoprotective property of prostaglandins is also fulfilled by increased endogenous bFGF. In the retina, VEGF is expressed by retinal glial cells, including Muller cells, astrocytes, and microglia.22 Among human retinal

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cells, Miiller cells were found to have the most VEGF immunoreactivity.23 VEGF is involved in the development of vasculature in normal retina.22 Clinical studies have revealed a close correlation between active neovascularization induced by hypoxic injury and elevated VEGF concentrations in patients with diabetes mellitus, central retinal vein occlusion, and retinopathy of prematurity,24 suggesting a role for VEGF as an important angiogenic factor in these diseases. Experimental data from a mouse model of proliferative retinopathy showed a marked increase in VEGF expression in Miiller cells before the development of neovascularization, 25 and this neovascularization was supressed by soluble VEGF-receptor chimeric proteins that inhibited VEGF,26 indicating the involvement of Miiller cells and VEGF in neovascularization in proliferative retinopathy. bFGF is one member of a family of nine heparin-binding proteins, thought to play key roles in angiogenesis.27'28 It is also known that bFGF exerts neurotrophic actions on a variety of neurons. 2930 Our previous studies 31 " 33 have revealed that, in the Royal College of Surgeons rat with an inherited photoreceptor degeneration and in constant light-induced photoreceptor degeneration, intraocular injection of bFGF protected photoreceptors. More recently, it has been demonstrated that mechanical injury to the retina induced a substantial increase in bFGF expression. This elevation was concentrated in the vicinity of the lesion and localized mostly to the inner nuclear layer in which Miiller cell bodies reside.34 We have also shown in a previous study35 that bFGF induces endogenous bFGF gene expression in cultured rat Miiller cells through activation of the protein kinase C pathway. Our findings indicate that Miiller cells may respond to injury by producing such growth factors as bFGF, and the stimulated production of these factors may be responsible for the photoreceptor protection in the presence of injury. Recent studies showed that PGE2 stimulated VEGF expression in cultured osteoblast cells 21 and synovial fibroblasts,36 providing evidence that PGE2 was capable of inducing VEGF expression. To the best of our knowledge, there is no such report in retinal Miiller cells that PGE2 induces VEGF and bFGF gene expression. The importance of Miiller cells in producing VEGF and bFGF and the involvement of prostaglandins in neovascularization and cytoprotection motivated us to use rat Miiller cells in culture as a model system to explore the possible role of prostaglandins in regulating VEGF and bFGF mRNA expression. We found that PGE2 induced VEGF and bFGF expression in cultured Miiller cells in a dose- and time-dependent fashion, whereas other inflammatory mediators, such as bradykinin, histamine, substance P, and vasoactive intestinal peptide, had no effect on VEGF and bFGF mRNA expression. Our results also provide evidence that the induction of VEGF by PGE2 was mediated by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and that the induction of bFGF by PGE2 was through the activation of pathways for PKA and protein kinase C (PKC). These findings raise the possibility that endogenous PGE2 stimulates VEGF and bFGF mRNA expression in Miiller cells in vivo under conditions in which production of PGE2 is increased, such as injury.

MATERIALS AND METHODS

Animals and Cell Culture All animals used in this study were cared for and handled according to the tenets of the ARVO Statement for the Use of

IOVS, March 1998, Vol. 39, No. 3 Animals in Ophthalmic and Vision Research. Miiller cells were cultured and identified as described previously. 35 Briefly, eyes from Sprague-Dawley rats of postnatal days 1 though 3 were enucleated and placed in essential modified Eagle's medium at room temperature overnight before transfer to a digesting medium (essential modified Eagle's medium containing 0.1% trypsin and 70 U/ml collagenase) for 30 minutes at 37°C. Retinas were gently dissected under a microscope. Cells were dissociated by trituration with a sterile Pasteur pipet, seeded into 10-cm culture dishes (Falcon, Oxnard, CA) containing growth medium (essential modified Eagle's medium, 10% fetal calf serum, 2 mM glutamine, and 1:1000 penicillin-streptomycin), and cultured at 37°C in a 5% CO 2 -95% air atmosphere in a humidified incubator. Neuronal cells and retinal debris were removed by forcibly pipetting the medium onto the culture dish three to five times when primary culture reached semiconfluency (5-7 days). Confluent cultures were passaged no more than four times. Cells were identified by immunocytochemical analysis using antibodies against the Miiller cell markers, including vimentin, carbonic anhydrase II, and glutamine synthetase. Cells were treated with PGE2, PKA inhibitors H-89 or SQ 22536, PKC inhibitors calphostin C or GF 109203X, PKC activator PMA, or the PKA activator forskolin. PGE2 and forskolin were from Sigma Chemical (St. Louis, MO); H-89, PMA, calphostin C, GF 109203X, and SQ 22536 were from Biomol (Plymouth Meeting, PA). We used phosphate-buffered saline for dissolving PGE2, forskolin, and SQ 22536 and used dimethyl sulfoxide solution for H-89, calphostin C, and GF 109203X. Addition of phosphate-buffered saline or dimethyl sulfoxide alone to the culture medium did not alter VEGF and bFGF mRNA levels.

RNA Extraction and Northern Blot Analysis RNA extraction and northern blot analysis were performed as described previously. 35 Cultured rat Miiller cells were lysed and homogenized in 5 5 M guanidinium thiocyanate solution. Total RNA was isolated using a cesium trifluoroacetate gradient method (CsTFA; Pharmacia, Piscataway, NJ). 37 Twenty micrograms total RNA from each sample was electrophoresed on 1% agarose formaldehyde gels and transferred to a nylon membrane (Hybond-N; Amersham, Arlington Heights, IL). Blots were UV irradiated to immobilize RNA and prehybridized for 4 hours at 50°C. Random-primed, 32P-labeled cDNA probes (VEGF, 930-bp human VEGF cDNA, gift of N. Ferrara38; bFGF, 477-bp rat bFGF cDNA, gift of A. D. Baird39; and 18S rRNA, 1.1-kb cDNA, gift of D. Schlessinger40) were added to the hybridization buffer (10 6 cpm/ml) and hybridized at 50°C overnight. After the posthybridization wash, the blots were exposed to a storage phosphor screen (Molecular Dynamics, Sunnyvale, CA), and the data were digitized by scanning the phosphor screen with a phosphor imaging system (Molecular Dynamics). In all northern blots, VEGF mRNA was detected as a single band at approximately 3-8 kb, and bFGF mRNA was detected as a major band at approximately 7.0 kb, along with several minor bands at lower molecular weights. Data were digitized from this single band and analyzed using a phosphor imaging system (Molecular Dynamics). Hard copies of blots were obtained by exposing the blots to film (Hyper Film; Amersham). The blots were reprobed with the 18S rRNA probe, and the data from the 18S rRNA served as a control for RNA loading.

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PGE2 concentrations (u.M) FIGURE 1. Dose-response effects of prostaglandin E2 (PGE2) on the induction of vascular endothelial growth factor (VEGF) mRNA expression. (A) Muller cells were exposed to different concentrations of PGE2 for 2 hours. The cultures without PGE2 treatment served as the control. A major VEGF transcript was detected in all lanes (indicated at the left). Migration of 28S and 18S rRNA is indicated at the right (upper panel)- The same blot was stripped of the VEGF probes and rehybridized with probes for 18S rRNA, which served as a the control for RNA loading (lowerpanel)- The concentration of PGE2 is indicated at the top of each lane. (B) Data from three independent experiments were averaged and presented as relative to the control level (mean ± SD, n = 3). *P < 0.05; **P < 0.01 versus the control.

Statistical Analysis Statistical differences were evaluated by one-way analysis of variance with the Newman-Keul's test for significance.

RESULTS In early experiments, we examined the effect of the inflammatory mediators, PGE2 (10 jxM), bradykinin (10 /xM), histamine (10 ju,M), substance P (10 /xM), and vasoactive intestinal peptide (10 /u-M), on VEGF and bFGF gene expression in cultured rat Muller cells. Among these mediators, only PGE2 induced VEGF and bFGF mRNA expression when added to the culture medium (data not shown). Further experiments revealed that PGE2 induced VEGF and bFGF gene expression in cultured rat Muller cells in a dose- and time-dependent manner.

Prostaglandin E2 Induces Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor mRNA Expression VEGF mRNA was detected as a band of approximately 3 8 kb (Fig. 1 A) in all samples. Figure 1 shows VEGF mRNA expression when cells were stimulated with four concentrations of PGE2 for 2 hours. Quantitative data from three independent experiments are shown in Figure IB. An increase in VEGF mRNA of 1.5-fold was observed at 0.1 /xM PGE2. The increase was approximately 1.8-fold at 1 /xM and reached a maximum of 2-fold at 10 JLIM. No further increase was observed at 100 fxM. PGE2. Figure 2A, shows the time course of VEGF mRNA expression induced by PGE2. When cells were treated with 10 /u,M PGE2, induction of VEGF mRNA expression was seen as early

as 0.5 hours. It increased to 1.7-fold by 1 hour, reached a maximum of more than 2.3-fold by 2 hours, and then slowly declined to the baseline level by 24 hours. The expression of bFGF mRNA (as 7.0-kb bands in northern blots) was relatively low in the control cells (Fig. 3). Induction of bFGF mRNA expression was first observed at a PGE2 concentration of 0.1 ju.M. The maximal induction was reached (3.5-fold) at 10 /xM of PGE2. A further increase in PGE2 concentration (100 /AM) did not increase bFGF mRNA. Induction of bFGF mRNA expression by PGE2 was time dependent, (Figs. 4A, 4B). With 10 /xM PGE2, induction was seen as early as 1 hour after PGE2 treatment. It reached a maximum of more than 3.5-fold at 2 hours and then slowly declined to the baseline level by 24 hours (Fig. 4A).

Induction of Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor mRNA Expression by Prostaglandin E2 through the Protein Kinase A Pathway We used the adenylate cyclase activator forskolin, the selective PKA inhibitor H-89, and the adenylate cyclase inhibitor SQ 22536 to determine whether PGE2-stimulated VEGF and bFGF mRNA expression are mediated by PKA in Muller cells. As shown in Figure 5, forskolin (10 jxM, 2 hours) induced a 1.7-fold increase in VEGF mRNA (Fig. 5A; lane 2), and PGE2 (10 /xM, 2 hours) stimulated a more than 2-fold increase in VEGF mRNA. Treatment with forskolin (10 jaM, 2 hours) plus PGE2 (10 ju,Mt 2 hours) resulted in no additional increase in VEGF mRNA (Fig. 5A, lane 4), suggesting that PGE2 and forskolin induced VEGF mRNA expression through the same pathway. However, forskolin (10 JLLM, 2 hours) induced a 2.9-fold increase in bFGF mRNA (Fig. 6A, lane 3).

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FIGURE 2. Temporal expression of vascular endothelial growth factor (VEGF) mRNA in response to prostaglandin E2 (PGE2). (A) Miiller cells were exposed to PGE2 (10 fiM) at different times in hours. Time after PGE2 treatment is indicated at the top of each lane. (B) Data from three independent experiments were averaged and compared with the control level (mean ± SD, n = 3). *P < 0.05; **P < 0.01 versus the control. Treatment with forskolin (10 /u,M, 2 hours) and PGE2 (10 fiM, 2 hours) resulted in an enhanced effect on bFGF mRNA expression (Fig. 6A; lane 4). Pretreatment with H^89 (30 JLIM, 1 hour before adding PGE2) inhibited the induction of VEGF (Fig. 5A; lane 6) and bFGF (Fig. 7A; lane 4) mRNA expression. Treating cells with H-89 (30 juM) alone for 3 hours had no effect on the baseline level of VEGF (Fig. 5A; lane 5) and bFGF (Fig. 7A; lane 3) mRNA. In addition, SQ 22536 inhibited VEGF and bFGF mRNA production by PGE2 (Figs. 8, 9). SQ 22536 is a cell-permeable,

adenylate cyclase inhibitor used in biologic systems to demonstrate that prostaglandins stimulate adenylate cyclase activity.'" When the cells were pretreated with different concentrations of SQ 22536 (100, 500, and 1000 ixM) for 1 hour, the PGE2-induced VEGF and bFGF mRNA expressions were inhibited in a dosedependent manner. SQ 22536 (1000 joM) alone had no affect on the baseline level of VEGF and bFGF mRNA (Figs. 8, 9). These results indicate that the induction of VEGF and bFGF mRNA expression by PGE2 occurs through the adenylate cyclase-protein kinase A pathway in cultured Miiller cells.

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PGE2 concentration (|iM) FIGURE 3. Dose-response effects of prostaglandin E2 (PGE2) on the induction of basic fibroblast growth factor (bFGF) mRNA expression. (A) Miilier cells were exposed to different concentrations of PGE2 for 2 hours. The cultures without PGE2 treatment served as the control. A major bFGF transcript was detected in all lanes (indicated at the left). Migration of 28S and 18S rRNA is indicated at the right (upper panel). The same blot was stripped of the bFGF probes and rehybridized with probes for 18S rRNA, which served as a control for RNA loading (lower panel). The concentration of PGE2 is indicated at the top of each lane. (B) Data from three independent experiments were averaged and compared with the control level (mean ± SD, n = 3). *P < 0.05; **P < 0.01 versus the control.

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Temporal expression of basicfibroblastgrowth factor (bFGF) mRNA in response to prostaglandin E2 (PGE2). (A) Muller cells were exposed to PGE2 (10 /xM) at different times in hours. Time after PGE2 treatment is indicated at the top of each lane. (B) Data from three independent experiments were averaged and compared with the control level (mean ± SD, n - 3). *P < 0.05; **P < 0.01 versus the control.

Protein Kinase C and Prostaglandin E2-Induced Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor mRNA Expression We next investigated the possible involvement of PKC in PGE2-induced VEGF and bFGF mRNA expression in cultured Muller cells. PMA is a PKC activator, although prolonged exposure to PMA downregulates intracellular PKC activity.

PMA treatment (0.2 /xM, 2 hours) induced a 6-fold increase in bFGF mRNA. Prolonged treatment with PMA (0.8 juM, 16 hours) blocked PGE2-induced bFGF mRNA expression (Fig. 10A) and induction of bFGF mRNA expression by PMA (Fig. 10A; lane 5). But downregulation of intracellular PKC by PMA (0.8 fiM, 16 hours) failed to show any inhibitory effect on PGE2-induced VEGF mRNA expression (Fig. 12A; lane 4).

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FIGURE 5. Effects of forskolin and the specific protein kinase A blocker H-89 on the induction of vascular endothelial growth factor (VEGF) mRNA. (A) Muller cells were treated with 10 pM forskolin (FKL) for 2 hours in the presence or absence of prostaglandin E2 (PGE2). H-89 (30 ju,M) was added to dishes 1 hour before PGE2 exposure and was observed for an additional 2 hours. In addition, cells were treated for 3 hours with 30 /xM H-89 or for 2 hours with PGE2 (10 juM), respectively. (B) Data from three independent experiments were averaged and compared with the control level (mean ± SD, n - 3). *P < 0.01 versus the control; +P < 0.01 versus the PGE2 group.

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Effect of forskolin on the induction of basic fibroblast growth factor (bFGF) mRNA expression. (A) Miiller cells were treated with 10 jiiM forskolin (FKL) for 2 hours in the presence or absence of prostaglandin E2 (PGE2). Cultures without treatment served as controls. (B) Data from three independent experiments were averaged and compared with the control level (mean ± SD, n — 3). *P < 0.01 versus the control.

We also used a specific PKC inhibitor, GF 109203X. Figure 11 shows that GF 109203X alone did not change the baseline level of bFGF mRNA (Fig. 11A; lane 6), but it blocked the effect of PGE2 on bFGF mRNA expression in a dosedependent manner when pretreating Miiller cells with dif-

ferent concentrations of GF 109203X (0.02, 0.2, and 2 fiM) for 1 hour before PGE2 treatment. In addition, pretreatment with GF 109203X (2 /LtM, 1 hour before adding PGE2) failed to inhibit the PGE2-induced VEGF mRNA expression (Fig. 12B). Pretreatment with calphostin C (1 /xM, 1 hour before

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7. Effect of protein kinase A inhibitor H-89 on the induction of basicfibroblastgrowth factor (bFGF) mRNA expression by prostaglandin E2 (PGE2). (A) Miiller cells were pretreated with 30 /xM H-89 for 1 hour and subsequently treated with 10 fxM PGE2 for 2 hours. Miiller cells were also treated with 30 /xM H-89 alone for 3 hours. (B) Data from three independent experiments were averaged and compared with the control level (mean ± SD, n = 3). *P < 0.01 versus the control; +P < 0.01 versus the PGE2 group. FIGURE

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+PGE2 FIGURE 8.

Effect of SQ 22536 on the induction of vascular endothelial growth factor (VEGF) mRNA expression by prostaglandin E2 (PGE2). (A) Miiller cells were pretreated with different concentrations of SQ 22536 (100, 500, and 1000 /nM) for 1 hour and subsequently treated with 10 juM PGE2 for 2 hours. In addition, Miiller cells were treated with 1000 yM SQ 22536 alone for 3 hours, PGE2 (10 /xM) alone for 2 hours. (B) Data from three independent experiments were averaged and compared with the control level (mean ± SD, n = 3). *P < 0.01 versus the control; +P < 0.05; + +P < 0.01 versus the PGE2 group.

adding PGE2), a specific PKC inhibitor that interacts with the PKC regulatory domain,42 did not block the PGE2-induced VEGF mRNA expression either (Fig. 12A; lane 6). These data suggest that the PKC pathway also is involved in PGE2-induced bFGF mRNA production but is not involved in VEGF mRNA expression.

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DISCUSSION We have shown that, in cultured rat Miiller cells, PGE2 induces the expression of VEGF and bFGF mRNA in a dose- and timedependent fashion, but no such effect was seen with other inflammatory mediators, such as bradykinin, histamine, sub-

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SJ 2.5-1 galactosamine, carbon tetrachloride, ethanol, and acetaminophen,59'60 and they are protective in experimental pancreatitis and myocardial ischemia.61'62 In addition, it has been found that PGE2 affords cytoprotection to embryonal neuroectodermal tissue and embryonic neural retina cells from degeneration induced by actinomycin C.18'20 More recently, a study63 revealed that increased endogenous production of PGE2, achieved by liposome-mediated combinant gene transfer of prostaglandin G and H synthase, protects rabbit lungs from endotoxin injury. The mechanisms that underlie cytoprotection have not been identified. Because of its well-known cytoprotective effects of bFGF, our finding, that PGE2 induces bFGF expression, may provide insight into the mechanism of prostaglandin-induced cytoprotection. We have reported that PGE2 stimulates the expression of VEGF, an important angiogenic factor, in cultured rat Muller cells. Because bFGF is thought to be involved in angiogenesis, our finding that PGE2 induces bFGF expression also suggests that PGE2 has a role in inducing neovascularization in the retina.

Acknowledgments The authors thank M. T. Matthes, D. Yasumura, and J. M. Wu for their assistance.

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