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(Investigative Ophthalmology and Visual Science. 1999;40:2945-2951.)
© 1999 by The Association for Research in Vision and Ophthalmology, Inc.

Release of bFGF, an Endothelial Cell Survival Factor, by Osmotic Shock

M. Elizabeth Hartnett1,2,3, Claudia M. Garcia2,3 and Patricia A. D’Amore1,2,3

1 From the Schepens Eye Research Institute, 2 Children’s Hospital of Boston, and 3 Harvard Medical School, Boston, Massachusetts.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To test the effects of osmotic change on basic fibroblast growth factor (bFGF) release from cultured endothelial cells (ECs).

METHODS. Bovine aortic and bovine retinal ECs were exposed to hypoosmotic shock for 2 minutes, were allowed to recover for 15 minutes, and had bFGF release assayed. The role of bFGF in cell recovery was assessed by including neutralizing antibody against bFGF or the addition of exogenous bFGF. Cell number and viability were determined under varying conditions. Apoptosis was assessed by immunoperoxidase detection of digoxigenin-labeled DNA.

RESULTS. After shock and recovery, both ECs released significantly greater amounts of bFGF than untreated control. bFGF release after shock for 2 minutes was lower than release after shock and recovery. Bovine retinal endothelial (BRE) cell number was reduced at 48 hours after shock, recovery, and removal of released bFGF compared with cells left in the presence of released bFGF. Cell number was significantly lower when BRE cells were shocked and recovered in the presence of a neutralizing anti-bFGF antibody (P < 0.05). Exogenous bFGF reversed this effect. Apoptosis was significantly increased in BRE cells shocked and recovered or in the presence of bFGF antibody (P < 0.001).

CONCLUSIONS. bFGF is released by cultured ECs in response to osmotically induced cell injury. These results support the concept of bFGF as a "wound" hormone and survival factor for ECs. In further compromised tissue, release of bFGF in this manner may play a role in the pathogenesis of disease.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Basic fibroblast growth factor (bFGF) is an important regulator of endothelial cell (EC) proliferation, migration, and protease production.1 2 3 bFGF does not have a consensus signal sequence,4 and, thus, the mechanism of its release is not well understood. One line of thinking holds that bFGF may be released via disruption of the cell membrane that occurs during nonlethal cell injury.5

This concept is supported by observations that cells survive disruptions of their plasma membranes caused by microneedle penetrations,6 7 scraping,8 and other mechanical pressures.9 10 For example, eccentric exercise of rat triceps revealed a significant increase in microscopically wounded cells.9 Rodent gut, injured by mechanical brushing of the luminal surfaces or by massage such that the luminal surfaces slid on one another while in the presence of fluorescein dextran, showed significant intracellular fluorescence, suggesting that cell membranes had been injured and resealed.10 In electrically stimulated ventricular myocytes, intracellular fluorescein dextran labeling indicated transient cell injury and resealing. These studies also identified bFGF as a trophic factor that leads to hypertrophy of the stimulated myocytes.11 In addition, both acidic fibroblast growth factor (aFGF) and bFGF were released from an ex vivo beating heart, suggesting that their release by contraction-induced wounding was an important mechanism of autocrine growth–promotion.12

Mechanical wounding of ECs in culture by scraping led to the release of bFGF.8 13 Also in cultured ECs, bFGF was found to associate with the cell surface and/or extracellular matrix where it was suspected to exert a paracrine growth effect on neighboring cells.14 Injury of rat smooth muscle cells in culture led to the release of bFGF, which then stimulated DNA synthesis of neighboring smooth muscle cells.15 Collectively, these observations have led to the concept of bFGF as a wound hormone.3 16

A growing body of work suggests that one or more of the FGFs may be important as survival factors. Cultured vascular ECs deprived of bFGF have been shown to undergo apoptosis.17 Inhibition of endogenous bFGF by antisense RNA induced apoptosis in vascular smooth muscle cells in vitro, and this could be prevented by administration of exogenous bFGF.18 Furthermore, bFGF inhibited apoptosis of cultured adherent human umbilical vein endothelial cells.19 An antisense oligonucleotide against aFGF prevented the induction of tumor necrosis factor-{alpha}–induced aFGF expression, which led to apoptosis in a spontaneously transformed human umbilical vein cell line.20 In retinal pigment epithelial (RPE) cells depleted of serum, the addition of bFGF reduced apoptosis and led to an increase in the synthesis and secretion of aFGF.21

We tested whether bFGF is released by ECs in vitro in response to changes in cellular osmolarity. Repeated variation in osmolarity may lead to cell injury such as what might occur in small capillaries in diabetes mellitus or other conditions of poor vascular flow. Release of bFGF by this mechanism may be relevant to the survival and proliferation of injured or neighboring cells as well as the induction or amplification of neovascularization seen in proliferative retinopathy.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell Cultures
Bovine aortic endothelial cells (BAEs)22 and bovine retinal endothelial cells (BREs)23 were maintained at 37°C as indicated: BAE in Dulbecco’s modified Eagle’s medium (DMEM), 10% calf serum, 10% CO2, and BRE in DMEM, 5% calf serum, and 5% CO2. All media were supplemented with glutamine (0.292 mg/ml), penicillin (100 U/ml), and streptomycin C (100 µg/ml; Irvine Scientific, Santa Ana, CA). All cells were used between passages 6 and 20.

Hypoosmotic Shock and Recovery
BAEs and BREs were plated at 5 x 104 cells/cm2. Cells were grown under conditions specified above for 3 to 4 days until 50% to 70% confluence. Hypoosmotic shock and recovery were originally designed to incorporate aequorin into mammalian cells, permitting the detection of a calcium-dependent signal while leaving the cells intact and viable.24 These observations indicated that this procedure caused microdisruptions in the plasma membrane without causing cell death. For hypoosmotic shock and recovery, cells were exposed to hypoosmotic shock (shocked) with 1.0 ml of 3 mM HEPES (3–4 mOsm/l) for 2 minutes, followed by the addition of 72.5 µl 2 M KCl in 3 mM HEPES for 15 minutes (recovered). Addition of the recovery solution returned the overall osmolarity to the normal range, approximately 250 mOsm/l. Heparin (10 µg/ml; Hepar Industries, Franklin, OH) was added to the cells with the shock solution to displace bFGF from the cell surface and extracellular matrix heparan sulfate for later collection and assay. The cells were examined by phase microscopy for changes in cell morphology.

Immediately after shock and recovery, the media were removed and additional media were added. In some cases, the original shock and recovery solutions were not removed, but additional media were added to determine the effect of retaining released bFGF on injured ECs. For controls, cells were exposed to media alone or to a mixture of shock (1 ml) and recovery solutions (72.5 µl) in the presence of 10 µg/ml heparin for 17 minutes.

Two methods were used to assess maximal bFGF release by cell injury. Cells were scraped in the presence of 10 µg/ml heparin and media8 or were exposed to two cycles of freezing and thawing over a dry ice/absolute alcohol mash.13

Assessment of Membrane Integrity
To determine whether plasma membranes were transiently injured by hypoosmotic shock and recovery, 1 to 10 mg/ml fluorescein–labeled dextran (11,000 and 20,000 MW; Sigma, St. Louis, MO) was included in the shock and/or recovery solutions. Cells were thus exposed to 2 minutes shock and 15 minutes recovery as indicated above while in the presence of fluorescein labeled dextran. The cells were then rinsed and refed with fresh media without serum or labeled dextran. The cells were immediately viewed and photographed under phase and fluorescence microscopy.

Analysis of bFGF
Directly after shock and recovery, the media were removed, clarified by centrifugation, and stored at -20°C, until assayed for bFGF. The amount of bFGF protein was determined using a bFGF ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The minimum detectable bFGF was 0.28 pg/ml as determined by the assay. Assays were performed in triplicate. bFGF values were normalized to 1 x 106 cells.

Analysis of Cell Number and Viability
Cell viability was assessed immediately after shock and recovery, up to 72 hours later. The cells were rinsed with phosphate-buffered saline and trypsinized in the presence of trypan blue added to media to yield an 0.08% final concentration. An aliquot was removed to determine cell number using a Coulter counter (Coulter Electronics, Hialeah, FL). Cell viability was determined by counting cells using a hemacytometer (American Optical, Buffalo, NY), considering cells permeable to the dye as nonviable. Control studies indicated that trypsinization did not reduce viability in cells that had been osmotically treated compared with controls (data not shown).

Effect of bFGF on Cell Survival and Proliferation
In some experiments after shock and recovery media were removed and replaced with fresh media containing 1:500 neutralizing sheep polyclonal anti-human recombinant bFGF (kindly provided by Michael Klagsbrun, PhD, Children’s Hospital, Boston, MA) or human recombinant bFGF (Scios Inc., Mountain View, CA; 2.5 ng/ml). Cell count and viability were determined at 6, 24, 48, or 72 hours.

Assessment of Apoptosis
Apoptosis in treated and control BREs was assessed by TdT-dUTP terminal nick-end labeling (TUNEL) assay, indicative of programmed cell death,25 as specified by the manufacturer (ApopTag; Oncor, Gaithersburg, MD). BREs were grown on four-well Labtek slides (2 cm2/well; Nalge Nunc International, Naperville, IL), shocked, recovered, and refed with fresh media. In some experiments, BREs were shocked in the presence of 1:500 bFGF antibody, recovered, and exposed to fresh media with 1:500 bFGF antibody. Results were compared with BREs that were shocked and recovered without removal of solution but rather addition of fresh media, and to shocked and recovered BREs with removal of solution and addition of media and 2.5 ng/ml bFGF. Assays were performed at 3 and 6 hours. These time points were based on the 4-hour peak of cell death with DNA fragmentation in human umbilical vein endothelial cells grown in media free of FGF.17 We assumed that if hypoosmotic shock and recovery with release of bFGF caused apoptosis in ECs, then the process of programmed cell death would begin immediately after this cell injury. An overall ANOVA was generated with subgroup analyses of Tukey.

Cells were fixed in 4% paraformaldehyde and assessed for apoptosis by TUNEL assay using the Apoptag kit. The localized horseradish peroxidase generated an intense signal with diaminobenzidene. Counter staining was performed using hematoxylin and eosin. Slides were viewed under 25 to 40x light microscopy. Brown nuclei were considered positive. Positive controls were cells treated with DNase 1 or 10 µm sections of bovine gut. Negative controls were unshocked BRE or BRE processed without exposure to TdT enzyme.

Statistical Analysis
In each experiment, three to four assays were performed for each condition and control. Mean values and standard deviations were determined. Statistical analysis was assessed by ANOVA with subgroup analysis (Tukey) for multiple comparisons or by Student’s t-test for two comparisons. Each experiment was repeated at least three times. Representative experiments described.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Effect of Hypoosmotic Shock on bFGF Release
Scraping BAEs leads to intracellular labeling with fluorescein–dextran and to the release of intracellular bFGF (80%) into the media.8 To determine whether hypoosmotic shock induces damage that can cause bFGF release, BAEs were exposed to hypoosmotic shock and recovered in the presence of fluorescein-labeled dextran. There was no labeling in control cells exposed to media. There was labeling of exposed cells but no significant difference in the percentage of fluorescently labeled cells whether fluorescein-labeled dextran was present during the shock and recovery periods or only during the recovery period (data not shown).

Hypoosmotic shock and recovery led to the release of bFGF by both types of EC examined. The absolute amounts of bFGF released varied among experiments compared with their controls. Shocked and recovered BREs released greater amounts of bFGF than did BAEs. Treated BAEs released 3- to 6-fold bFGF (i.e., 76.94 pg/106 cells compared with 25.21 pg/106 for control; Fig. 1 A, P = 0.001), and BREs released 5.5- to 6-fold bFGF (i.e., 886.29 pg/106 cells compared with 161.23 pg/106 for control; Fig. 1B , P = 0.0001). The average percentage of bFGF released by shock and recovery compared with that released by scrape injury was 20% for BAEs and 39% for BREs.



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  		Figure 1. Effect of hypoosmotic shock and recovery on bFGF release from ECs. BAE (A, *P = 0.001) and BRE (B, *P = 0.0001) were subjected to 2 minutes hypoosmotic shock and 15 minutes recovery. Control cells were in media or a mixture of shock and recovery solutions (250 mOsm/l). All counts done in triplicate; experiments were repeated three times.

 
To determine whether the bFGF release was due to cell death after shock and recovery, BAEs were exposed to hypoosmotic shock for 17 minutes versus shock for 2 minutes. After 17 minutes of hypoosmotic shock with no recovery period, bFGF released was 80% to 90% of that released after hypoosmotic shock and recovery. In contrast, however, cell viability of cells shocked for 17 minutes was 20% to 25% compared with 82% to 95% for cells shocked and recovered (control viability 85%–99%). These findings provide support that bFGF release is not the result of cell death.

To determine whether bFGF was released by hypoosmotic shock or whether the release was dependent on the recovery phase, bFGF released from ECs shocked for 2 minutes was compared with bFGF released from cells shocked for 2 minutes and recovered for 15 minutes. Release of bFGF after exposure to shock alone varied between the cell types (Figure 2) . Compared with bFGF levels released by cells shocked and recovered, release by cells only shocked was significantly less for BREs. This difference was not found for BAEs. These results indicate that the recovery phase is important to the release of bFGF for BREs.



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  		Figure 2. Comparison of hypoosmotic shock and recovery versus hypoosmotic shock alone on bFGF from ECs. BAEs (A) and BREs (B) subjected to 2 minutes of hypoosmotic shock and 15 minutes of recovery versus ECs exposed to 2 minutes of hypoosmotic shock alone. BAE hypoosmotic shock and recovery versus shock alone (column 1 versus 2; *P = 0.65). BRE hypoosmotic shock and recovery versus shock alone (column 1 versus 2; *P = 0.003). Control cells were in media or a mixture of shock and recovery solutions (250 mOsm/l). All counts done in triplicate; experiments were repeated three times.

 
Effect of Hypoosmotic Shock and Recovery on Cell Viability
Viability of BAEs was assessed immediately after shock and recovery or at varying times after the cells were refed with fresh media. The viability of BAEs immediately after hypoosmotic shock and recovery ranged from 73% to 89%, compared with control 87% to 96%; for example, 4.03 ± 0.88 x 105 immediately after shock and recovery (viability 87%) versus 4.73 ± 0.67 x 105 control (viability 89%). Six hours after removal of the shock and recovery media and the addition of fresh media, cell viabilities were similar to those of control cells (Table 1) . BAE number at 6 and 24 hours after shock and recovery and replacement of fresh media was similar to control cell counts (compare 3.66 x 105 at 24 hours after shock and recovery with 3.87 x 105 control).


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  		Table 1. BAE Viability after Shock and Recovery

 
After 48 hours in fresh media, BRE shocked and recovered displayed 83% viability compared with 87% in control cells, and at 72 hours viability of shocked and recovered BREs was 89% compared with 96% in control (Table 2) . When the media, and thus released bFGF, were left in place for 48 hours after shock and recovery, BRE number approached control counts (1.77 ± 0.38 x 105 versus 1.85 ± 0.08 x 105; P = NS), whereas when the media (and, thus, released bFGF) were removed, the number of BREs that were exposed to shock and recovery was lower at 48 hours, although not significantly (1.47 ± 0.04 x 105 versus 1.85 ± 0.08 x 105; P = NS). At 72 hours, BRE counts (viability) were 2.87 ± 0.33 x 105 (89%) for shocked and recovered cells with removal of media and released bFGF versus 2.27 ± 0.23 x 105 (96%) control. Despite variability among experiments, the overall results suggest that released bFGF after injury may be necessary, although not necessarily sufficient, for subsequent cell viability and proliferation.


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  		Table 2. BRE Viability after Shock and Recovery

 
Effect of bFGF
To determine whether released bFGF was playing a role in cell recovery, a neutralizing polyclonal antiserum against bFGF was added to BREs that were shocked and recovered, and cell number and viability were determined 48 hours later. In these studies, the number of BREs was 43% lower at 48 hours than control cells (P < 0.05; ANOVA; Fig. 3 ). In contrast, BREs that were shocked and recovered and then cultured for 2 days in media with bFGF added (2.5 ng/ml) had a trend toward increased cell number compared with that of antibody-exposed cells (1.31 ± 0.36 x 105 versus 1.06 ± 0.09 x 105). However, the variability in cell numbers led this increase to be insignificant (Fig. 3) .



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  		Figure 3. Effect of bFGF neutralization and bFGF on BRE subjected to hypoosmotic shock and recovery. BRE subjected to 2 minutes of hypoosmotic shock and 15 minutes of recovery in the presence of bFGF or antibody to bFGF. Control represents cells not subjected to hypoosmotic shock and recovery, bFGF, or antibody to bFGF. Cell counts (Coulter counter) determined at 48 hours. Overall ANOVA was significant (P < 0.0001). Subgroup testing of control cells (column 1) versus shocked and recovered cells in the presence of bFGF antibody (column 5) was significant (P < 0.05). Control cells versus shocked and recovered cells with exogenous bFGF added (column 6) was not significant (P > 0.05). All counts done in triplicate; experiments were repeated three times.

 
Cells that were shocked and recovered in the presence or absence of bFGF antibody, exogenous bFGF, or fresh media were examined at 3 and 6 hours for cells undergoing apoptosis. The overall ANOVA was significant (P < 0.0001; see Table 3 ). The percentage of TUNEL-positive cells at 6 hours after shock, recovery, and removal of media was higher than control, untreated cells (13.4% ± 3.3% versus 0.6% ± 0.5%, P < 0.001 Tukey). However, the percentage of TUNEL-positive cells at 3 hours after shock, recovery, and removal of media was not significantly higher than control, untreated cells (5.6% ± 2.6% versus 0.2% ± 0.4%, P = NS). The percentage of TUNEL-positive cells after treatment was significantly higher after 6 hours than after 3 hours (P < 0.01). Cells shocked and recovered in the presence of bFGF antibody were also significantly more TUNEL-positive at both 3 hours (12.7% ± 3.5%) and 6 hours (13.0% ± 6.0%) compared with controls (P < 0.001). When media, and thus released bFGF, were not removed after shock and recovery, the percentage of TUNEL-positive cells was lower than when media were removed after shock and recovery. For example, at 6 hours after injury and removal of media, there were 13.4% TUNEL-positive cells versus 4.9% when media were not removed (P < 0.01). However, the difference at 3 hours was not significant (5.6% versus 4.8%). The addition of fresh media and bFGF led to a slight but not significant reduction in the number of TUNEL-positive cells at 3 hours (6.2% ± 5.6%) and 6 hours (6.8% ± 7.3%) compared with cells shocked and recovered with removal of media (see Table 3 ).


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  		Table 3. Percent Apoptosis in Treated BRE

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We found that when ECs were subjected to hypoosmotic shock and then recovered, they released significantly greater amounts of bFGF than control cells. For BREs, bFGF release depended on the recovery phase and not on shock alone. The amount of bFGF released was approximately 20% to 40% of that released by cells that were scrape-injured.8 The bFGF release appears to result from injury and resealing of the cell membrane, as evidenced by cytoplasmic labeling of shocked and recovered cells with fluorescein-labeled dextran, a molecule that is otherwise impermeable to the plasma membrane.

The released bFGF did not result from cell death alone. After 17 minutes of shock with no recovery, cell viability dropped dramatically (to 20%–25%); yet, the increased cell death did not result in a greater release of bFGF than that after shock and recovery. The fact that the amount of bFGF released by cells with greatly reduced viability was not greater than that released by cells recovered from hypoosmotic shock indicates that cell death is not a prerequisite for bFGF release. This agrees with other investigators who found that ECs grown under conditions that varied the strength of cell adherence after scrape injury and, thus, the cell viability, had a weak correlation between cell death and growth factor release.13

It appears that release of bFGF is not only dependent on hypoosmotic shock but also on the recovery phase. One possible explanation for the increase in bFGF released during the recovery phase is that as the cell is brought into a near-normal osmotic environment after hypoosmolar shock, the cell membrane may undergo perturbations that may lead to further release of bFGF. Clinically, large changes in the osmolarity of the cell microenvironment may occur at the microvascular level. These would not be apparent in a serum measure of osmolarity. In an extreme clinical situation of osmolarity change (e.g., diabetic hyperosmolar coma), the average fluid deficit is 10 to 11 liters. Microvasculature flow is reduced substantially or even shut down. Microvascular osmolality could rise tremendously even when the serum osmolality may only reflect 380 mOsm/l, a measurement consistent with diabetic hyperosmolar coma.

Two lines of evidence suggest that bFGF released during cell injury is important to cell survival. When media (and, therefore, released bFGF) were removed from shocked and recovered BREs, cell numbers were decreased compared with control at 48 hours. There was also a trend for cells that remained in the presence of bFGF released by injury after shock and recovery to maintain cell counts comparable to control. These results suggest that released bFGF is important in recovery from cell injury and in cell survival. When released bFGF was neutralized by the addition of antisera, BRE number after 48 hours was 43% lower than control cells, whereas the addition of bFGF to shocked and recovered cells increased cell counts to 71% of control numbers.

Our observations, along with those of other investigators,8 11 12 15 indicate that bFGF is released during and as a result of cell injury. If the injury is not lethal, the released bFGF may be an important component of the healing process. One hypothesis is that release may occur as a result of daily function and "wear and tear" in the particular tissues. Studies on intestine,10 myocytes,9 11 and RPE cells26 support the hypothesis that injury associated with a cell’s specific function leads to bFGF release. For example, injury in the intestine occurs with peristaltic movement of food through the intestinal tract.10 In muscle, exercise leads to cell injury during contraction and elongation of myocytes. In the outer retina, cell injury may occur from light toxicity and associated oxidative stress.

The released bFGF then appears to be important for cell survival. In support of this, apoptosis has been induced by the inhibition of bFGF.17 18 Conversely, apoptosis was reduced in cultured RPE cells depleted of serum when bFGF was added.21 Rat smooth muscle cells injured with a soft plastic tube released bFGF, which activated DNA synthesis of neighboring cells both directly and via stimulation of platelet-derived growth factor (PDGF AA).15 Our data also suggest that bFGF released during a change in osmolarity acts in part as a survival factor. There were significantly more cells undergoing apoptosis at 6 hours after shock and recovery than control. We found that the neutralization of released bFGF led to significantly greater apoptosis (P < 0.001) compared with control cells at 3 and 6 hours. In addition, at 6 hours, the percentage of TUNEL-positive cells was significantly lower in shocked and recovered BREs when media and released bFGF were not removed compared with when shocked and recovered BREs had media removed (P < 0.01; see Table 3 , compare d versus j). When media were removed but fresh media and bFGF were added, there was a trend toward reduced apoptosis. This may suggest that endogenous material released may be more important as a survival factor for injured BREs than exogenously added bFGF.

bFGF’s role as a survival factor may have clinical importance. In the visual system, bFGF has been implicated as a trophic factor. When applied to transected optic nerve, the number of retinal ganglion cells surviving axotomy after 30 days tripled compared with control.27 bFGF injected into the subretinal space delayed photoreceptor degeneration in rats with inherited retinal dystrophy28 or phototoxicity.29 After optic nerve crush injury, bFGF expression was increased in the optic tract within days and in the photoreceptors several weeks after injury.31 In addition, transgenic mice in which dominant-negative FGF receptors were expressed under the control of the rhodopsin promoter developed photoreceptor damage, dropout, and retinal degeneration.30

Sharp changes in local osmolarity are clinically relevant. At the microvascular level, changes in osmolarity may fluctuate greatly in situations of hyperosmolarity or metabolic shifts, which are seen in vascular shunting or diabetes mellitus. These osmotic changes represent a specific form of cell injury for ECs. To survive, ECs may release bFGF that in turn affects injured and neighboring cells. Although in vitro studies suggest that BAE viability may not be adversely affected even at 460 mOsm/l,32 osmolarity fluctuations from either low-to-high levels or vice versa in vivo may differ from in vitro studies. Also, micro- or macrovascular EC may respond differently in vivo to osmolarity changes. Our study suggests that release of bFGF after recovery from hypoosmotic shock may provide a mechanism for continued cell survival.

In the presence of additional cell or tissue compromise, the released bFGF may play a role in pathologic change, inducing EC proliferation.1 2 3 Evidence suggests regulation between vascular endothelial growth factor (VEGF) and FGF signal transduction pathways in a model of RPE cells that overexpress VEGF.33 bFGF can induce VEGF expression in proliferating EC, but antibody to VEGF reduces bFGF-induced EC proliferation.34 This may be important in retinovascular disease associated with retinal ischemia, such as diabetic retinopathy, retinopathy of prematurity, or retinal vein occlusion, in which hypoxia-induced VEGF is associated with preretinal neovascularization.35 36 37 Either initial or subsequent release of bFGF related to tissue injury associated with disease processes may result in further VEGF expression and be additive or even synergistic38 to the pathologic neovascular response.


   Footnotes
 
Supported by NIH grant EY05985 (to PAD).

Submitted for publication November 20, 1998; revised May 17, 1999; accepted July 19, 1999.

Commercial relationships policy: N.

Corresponding author: M. Elizabeth Hartnett, Harvard Medical School, 20 Staniford Street, Boston, MA 02114. E-mail: hartnett{at}vision.eri.harvard.edu


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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