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Akt pathway
Experimental Eye Research 90 (2010) 726e733
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Pigment epithelium-derived factor inhibits erythropoietin-induced retinal endothelial cell angiogenesis by suppression of PI3K/Akt pathway Ravinarayanan Haribalaganesh, Sardarpasha Sheikpranbabu, Elayappan Banumathi, Sangiliyandi Gurunathan*
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Department of Biotechnology, Division of Molecular and Cellular biology, Kalasalingam University (Kalasalingam Academy of Research and Education), Anand Nagar, Krishnankoil 626190, Tamilnadu, India
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Article history: Received 27 September 2009 Accepted in revised form 10 March 2010 Available online 16 March 2010
Erythropoietin (EPO) plays a critical role in the vascular system and exhibits angiogenic activity in endothelial cells (ECs) such as stimulation of cell proliferation, migration and tube formation in vitro. EPO is the major regulator of cell proliferation and differentiation of erythroid precursors and there by preventing the apoptosis. Pigment epithelial derived factor (PEDF) is a potent anti-angiogenic factor whose effects are partially mediated through the induction of EC apoptosis. The mechanism of EPO and PEDF in retinal neovascularization has not been well documented yet. The effect of EPO and PEDF on cell proliferation was determined by MTT assay. In vitro wound-scratch assay was performed to study the migration of ECs and in vitro tube formation was assessed by the on-gel assay system using gelatin. Inhibitor assay was carried out using LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Further, PI3K/Akt activity was assessed by transient transfection assay using constitutively active (CA) and dominant negative (DN) Akt mutants. Dextran permeability assay was performed to determine the vascular permeability. We report that EPO stimulates EC proliferation, migration, tube formation and permeability whereas PEDF inhibits the EPO-induced ECs proliferation and permeability. Over expression of DN Akt blocked EPO stimulation of proliferation and permeability, while over expression of CA Akt rescues the inhibitory effect of PEDF on proliferation and permeability. These results demonstrate that PEDF may inhibit the EPO-induced proliferation and permeability via PI3K/Akt-dependent pathway. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
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Keywords: endothelial cells erythropoietin pigment epithelium-derived factor proliferation vascular permeability
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a r t i c l e i n f o
Angiogenesis is thought to depend on a precise balance of positive and negative regulation. The neovascularization of the retina occurs predominantly via angiogenesis, which is the formation of functional blood vessels from a pre-existing vasculature (Ray and Thomas, 2005). Although this process was tightly regulated under physiological conditions, persistent unregulated angiogenesis is a hallmark of diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity and age-related macular degeneration. Diabetic retinopathy is one of the significant microvascular complications in diabetes and is a leading cause of acquired blindness among people of occupational age (Frank, 1991). The vascular permeability and its regulatory control are central to homeostasis and increases in vascular permeability play a key role in the development of sepsis-associated hypotension, acute
* Corresponding author. Tel.: þ91 4563 289042; fax: þ91 4563 289322. E-mail address: [email protected] (S. Gurunathan). 0014-4835/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2010.03.005
respiratory distress syndrome, nephrotic syndrome, diabetic retinopathy and diabetic neuropathy. Erythropoietin (EPO) is a 34-kDa renal glycoprotein hormone, responsible for the development of immature erythroid cells (Koury and Bondurant, 1992; Sarah et al., 1997) and was first characterized as a hematopoietic factor. More recently, EPO and EPO receptor (EPOR) were found to be expressed by brain and retinal tissue (Jelkmann and Metzen, 1996; Hugo et al., 2006; Juul et al., 1998; Böcker-Meffert et al., 2002). Besides an increase of intracellular calcium, the main signaling pathways of hematopoietic cells involved in the activation of phosphatidylinositol 3-kinase (PI3K/Akt), MAPK, JAK and activation of transcription factor 5 (STAT5) for survival, proliferation, and migration of stimulated cells (Raul et al., 1995; Domenico et al., 1999; Dolores et al., 2002). Pigment epithelium-derived factor (PEDF) is a 50-kDa secreted glycoprotein that was first isolated from conditioned medium of cultured primary human fetal retinal pigment epithelial cells (Tombran and Johnson, 1989). PEDF is widely expressed throughout fetal and adult tissues, including the adult brain (Bilak et al., 1999), spinal cord (Kuncl et al., 2002), plasma (Steen et al., 2003),
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2. Materials and methods 2.1. Cell culture
GRECs were seeded at a density of 2 103 cells per well into 96well culture plates. After attachment, the culture medium was changed to IMDM containing 10% FBS, and the cells were grown for 24 h. After reaching 80% confluence, the cells were starved with IMDM containing 1% FBS for 5 h and then treated with various concentrations of EPO (Sigma St Louis, USA) or PEDF (Abcam, Cambridge, UK) and/or combinations of both. After 24 h, cell proliferation was assessed using the MTT assay as per the manufacturer’s instructions (MTT-cell proliferation kit, Roche, Germany GmbH). Absorbance was measured at 595 nm using an enzymelinked immunosorbent assay (ELISA) plate reader (Biorad Model 680, Japan). 2.3. In vitro cell migration assay
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To examine the cell migration effect of EPO and PEDF on GRECs, a wound-scratch assay was followed as described previously (Mary et al., 2005). Cells were seeded in 35-mm plates and grown to confluence in IMDM growth medium. The monolayer was starved for 5 h in IMDM with 1% FBS. Cells were treated with 1 mM 5-Fluorouracil (Sigma St Louis, MO) for 15 min to rule out the contribution of differences in cell proliferation. Endothelial cells were aseptically scraped out from the middle of the plate using a sterile 200-ml micropipette tip rinsed with growth medium. After scraping, cells were rinsed twice with medium to remove woundderived cells. Then the cells were scratched with a plastic pipette tip to yield cell-free areas of uniform width and treated with EPO and PEDF. Control plates contained pure assay medium (1% serum, without growth factors). After incubation at 37 C for time intervals of 0 and 24 h, movement of cells into the denuded area was evaluated. To determine the extent of wound closure, digital photographs were taken (two pictures from each plate at randomly chosen areas) after 24 h and the wound closer area was analyzed by axiovision software (Zeiss, Germany).
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liver (Sawant et al., 2004), bone (Gerald et al., 2005), eye, heart, and lung (Joyce et al., 1996). It is one of the most potent endogenous inhibitor of angiogenesis in the eye (Dawson et al., 1999; Veronica et al., 2001). Several studies have shown that a decreased level of PEDF in the eye is associated with a number of ocular neovascular and neurodegenerative diseases. Inhibition of vessel formation was mediated by up-regulation of the Fas-ligand on the surface of endothelial cells, with subsequent induction of apoptosis in actively dividing cells (Olga et al., 2002). PEDF selectively inhibits the formation of new vessels from endothelial cells but does not appear to harm the existing vascular structure. The inhibitory effect of PEDF on vessel formation appears to be reversible when a transient and regulated angiogenesis occurs in situations including tissue repair after injury. A recent study has provided clinical evidence to show that expression of VEGF and PEDF is inversely correlated during the development of retinal neovascularization, further supporting an anti-angiogenic role for PEDF in-vivo (Nahoko et al., 2002). Furthermore the high expression of EPO and low expression of PEDF has been established in angiogenesis but the correlation effect of the EPO and PEDF remains unclear. In the present study, we have investigated the molecular mechanism of PEDF on EPO-induced angiogenesis, and permeability in goat retinal microvascular endothelial cells (GRECs). We report that EPO increases the cell proliferation, migration, tube formation and permeability significantly in GRECs. Further, PEDF inhibits the EPOinduced via angiogenesis and permeability via PI3K/Akt-dependent pathway and this pathway might represent a potential target to inhibit the neovascularization process, which may lead to the development of novel anti-neovascular agents for therapeutic purposes.
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Goat retinal microvascular endothelial cells (GRECs) were isolated and cultured as described previously (Sheikpranbabu et al., 2009a). Briefly, freshly isolated goat retinas were washed twice with sterile 1 Phosphate Buffer Saline (PBS), pH 7.2 and the retinal layer was separated in to retinal tissues and blood vessels. The blood vessels were minced and mixed with 1 PBS, transferred to a 15 ml tube, centrifuged at 600 g for 5 min at room temperature and the supernatant discarded. The pellet containing the blood vessels was suspended in 3 ml of proteolytic enzyme cocktail containing 0.01 g collagenase type IV, 0.004 g Pronase and 0.004 g DNAse, mixed with 2 ml of 1 PBS (pH 7.2), and incubated at 37 C for 20e25 min. Subsequently the digested blood vessels were centrifuged at 600 g for 5 min at room temperature and the supernatant discarded. The pellet containing the blood vessels was resuspended in 1.5 ml of fresh Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal bovine serum (FBS) (Sera Laboratories International Ltd, UK), 100 U/ml Penicillin-G, 100 mg/ml Streptomycin and 250 mg/ml Amphotericin-B. The suspended blood vessels were seeded in 35-mm tissue culture dish pre-coated with 1% gelatin, incubated at 37 C in 5% CO2 atmosphere. After 12 h the unattached blood vessels and debris were removed and washed once with serum free IMDM followed by the addition of fresh medium supplemented with 10% FBS and the cultures returned to 37 C with 5% CO2. The medium was changed every 3 days and the cell growth was monitored under phase contrast microscope. The cells were used between passages 4 and 9. 2.2. In vitro cell proliferation assay To assess the cell proliferation, a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay was used. Briefly,
2.4. In vitro capillary morphogenesis assay
The isolated GRECs were seeded at 3 104 cells in 35-mm tissue culture plates pre-coated with 4% gelatin. After 24 h, the growth medium was replaced with IMDM medium containing 0.5% FBS and PEDF in the presence or absence of EPO in GRECs and the plates were maintained at 37 C with 5% CO2. The capillary tube formation was observed under phase contrast microscope (Zeiss Axiovert, Germany) and the tubular length was measured using axiovision software. 2.5. Pharmacological inhibitor assay GRECs were seeded at 2 103 cells per well in a 96-well plate pre-coated with 1% gelatin. After 24 h, the growth medium was replaced with IMDM containing 0.5% FBS. The cells were treated with PI3K specific pharmacological inhibitor, LY294002 (10 mM) for 15 min prior to EPO treatment. The cell proliferation rate was analyzed using MTT assay according to the manufacturer’s instructions and the cell proliferation was measured using a micro plate Reader (Biorad Model 680, Japan). 2.6. Transient transfection assay Constitutively active (myristoylated) Akt (CA Akt) and dominant negative (K179M) Akt (DN Akt) mutants were kindly provided by Dr. Kyung-Jin Lee (Department of Life Science, GIST, Korea). Transient transfection assay was carried out using the method described earlier (Haribalaganesh et al., 2009). Briefly, GRECs were transiently transfected using a nucleofection technique
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(Amaxa Biosystems, Koeln, Germany) and grown to 80% confluence in IMDM and the cells were harvested by trypsinization and centrifuged at 3000 rpm for 10 min. The pellet was resuspended in the nucleofector solution (Basic nucleofector kit, Amaxa Inc., Germany) to a final concentration of 4e5 105 cells/100 ml. At the time of transfection, 1e3 mg of plasmid DNA encoding green fluorescent protein (pmaxGFP), CA Akt or DN Akt was added along with a nucleofector solution and subjected to electroporation using a nucleofector device-II (Amaxa Biosystems, Koeln, Germany). After electroporation, transfected cells were resuspended in 35-mm plates containing 1 ml of prewarmed IMDM and incubated in 5% CO2 at 37 C. The transfection efficiency determined after cotransfection with a pmaxGFP plasmid (Amaxa Inc.) and cell viability determined by MTT assay were usually about 80e90%.
3. Results 3.1. EPO induces the cell proliferation in GRECs Recent reports suggest that erythropoietin is a potent angiogenic molecule which induces cell survival and proliferation in retinal endothelial cells (Rachel and Aly, 2007). To determine the effect of EPO on cell proliferation in GRECs, cells were exposed to various concentration of EPO for 24 h. The effect of EPO on endothelial cell proliferation was determined by MTT colorimetric assay. EPO-induced maximal cell proliferation at 1 IU compared to control (Fig. 1A). This mitogenic effect was gradually lost as the concentration of EPO was increased in the medium to a maximum of 10 IU. The data suggest that EPO-induced cell proliferation significantly in GRECs.
2.7. Culture in permeable insert systems
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Endothelial cell permeability of a confluent cell monolayer was determined by a fluorescence-based assay as reported previously (Sheikpranbabu et al., 2009b). Briefly confluent monolayer endothelial cells (ECs) were incubated with IMDM assay medium for 5 h containing only 1% FBS without any growth factors. Here, we used Rhodamine isothiocyanate (RITC)-dextran (Sigma St Louis, MO) with a molecular weight of 70-kDa, which corresponds to a diameter of approximately 6 nm. The ability of the dextran to pass from the upper chamber of the permeable insert system into the lower chamber was used to determine the leakage properties of the monolayer. More dextran in the lower chamber indicates higher permeability. This dextran flux was quantified by means of the fluorescence in the lower chamber. 10 mM of RITC was applied to the apical chamber of the transwell inserts with a confluent ECs monolayer. Permeability assays started 4 days after seeding, when a confluent monolayer of ECs was present. EPO was added for the designated times. Where applicable, PEDF was added 15 min prior to EPO treatment. In some experiments, LY294002, a PI3-kinase inhibitor was added to ECs cultures 15 min prior to growth factor addition. The media volumes used equalized fluid heights in the apical and basolateral chambers, so that only diffusive forces were involved in solute permeability. At the indicated times after treatment, 100 ml samples were taken from the basolateral chamber and placed in a 96-well plate (96-well, polystyrene, flat bottom, Corning, USA). A sample was taken from the apical chamber at the last time point; the amount of fluorescence in this chamber did not change significantly over the course of the experiment. Aliquots were quantified using a fluorescence multi-detection micro plate reader (Biotek FLx800, Vermont, USA) at an excitation/emission of 540/590 nm.
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2.8. Dextran endothelial cell permeability assay
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GRECs at passage five were seeded in IMDM coated with gelatin in porous membranes of permeable insert system (Transwell, Corning, USA, pore size 0.4 mm, growth area 0.33 cm2) with a seeding density of 30,000 cells/cm2. The medium was changed the next day, and every other day thereafter. 500-ml medium was also added in the lower chamber of the insert systems.
2.9. Statistical analysis All results are expressed as the means standard deviation (SD) values. The statistical significance was evaluated by using the paired two-tailed student’s t test. A P < 0.05 was considered to indicate a statistically significant difference.
Fig. 1. (A) Effect of EPO on GRECs survival: GRECs were treated with different concentrations of EPO. Where 1 IU EPO induce Maximum proliferation (0.114 0.010, P < 0.05, n ¼ 3), (b) Effect of PEDF on GRECs survival: GRECs were treated with different concentrations of PEDF. Where 1 nM PEDF sufficiently inhibited the cell survival (0.037 0.006, P < 0.05, n ¼ 3), (C) Effect of PEDF on EPO-induced GRECs survival: 1 IU EPO-induced GRECs were treated with 1 nM PEDF. Where 1 nM PEDF inhibited the cell survival (0.063 0.030, P < 0.05, n ¼ 3).
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3.3. PEDF blocks EPO-induced endothelial cell migration
To study the ability of EPO to induce EC permeability, GRECs were grown to confluence on transwell membrane filters and then treated with EPO at various concentrations. EPO-induced dosedependent increase in permeability of the endothelial cell monolayer which is shown by the relative transfer of RITC-labeled dextran in 12 h, where maximal permeability was induced by the treatment with 2 IU/ml of EPO (Fig. 5A). The effect of EPO on permeability persisted over 24 h duration of the experiment. 3.7. PEDF inhibits endothelial cell permeability in a dose-dependent manner PEDF is known to act on multiple cell types in addition to endothelial cells such as neurons, glial cells, microglia, and retinal pigment epithelium, but we were interested in examining whether PEDF has a direct effect on endothelial cells in the regulation of permeability. To determine the role of PEDF on endothelial cell permeability, we examined the diffusion of non-radioactively labeled dextran across a confluent endothelial cell monolayer. PEDF reduced the permeability to RITC-dextran compared with control significantly (Fig. 5B). The inhibitory effect was dosedependent, reaching a maximum at 10 nM and a very less significant degree of inhibition occurred at lower doses. In addition, the effect of PEDF on permeability persisted over the 24 h duration of the experiment.
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Angiogenesis involves a variety of parallel events, including matrix degradation, migration, proliferation and morphogenesis of endothelial cells. To know the effect of PEDF and EPO on cell migration, a wound-scratch assay was performed. The assay showed that EPO significantly increases migration of endothelial cells; PEDF was observed to inhibit the cell migration. Fig. 2A shows enhanced migration of GRECs and wound closure being completed almost by 24 h in 1 IU EPO-treated plates, whereas a significant area of the wound remaining uncovered in 1 nM PEDF-treated plates. The same inhibitory effect was observed in plates incubated with combinations of EPO and PEDF, which shows that PEDF blocks the EPO-induced cell migration. Fig. 2B shows the quantitative assessments of cell migration under various conditions.
3.6. EPO enhances endothelial cell permeability in GRECs
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PEDF is a well-known inhibitor of angiogenesis and it is involved in EPO-induced barrier dysfunction of microvascular endothelial cells. To confirm the effect of PEDF on GRECs, cells were exposed to various concentrations of PEDF and incubated for 24 h. Among various concentrations of PEDF tested, 1 nM PEDF significantly inhibits the cell proliferation in GRECs (Fig. 1B). Further, to analyze the counteractive effect of PEDF on EPO-induced cell proliferation, 1 nM of PEDF was added 15 min prior to EPO treatment. The result shows that pre-treatment of PEDF significantly blocked the EPOinduced cell proliferation in GRECs (Fig. 1C).
wild type GRECs (Fig. 4B). These results show that EPO could possibly induce the cell survival via PI3K/Akt-dependent pathway, and PEDF might suppress the EPO-induced PI3K/Akt activity in GRECs.
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3.2. PEDF inhibits the EPO-induced cell proliferation
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3.4. PEDF inhibits EPO-induced tube formation in GRECs
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The ability of endothelial cells to form capillary-like structures on an extracellular matrix can distinguish endothelial cells from other common contaminating cell types. To further characterize the effect of EPO and PEDF on angiogenesis, the effect of EPO and PEDF on endothelial tube formation was analyzed using capillary morphogenesis assay. Fig. 3A shows that GRECs treated with 1 IU EPO organize well on the 4% gelatin and form extensive capillary networks. In contrast, the addition of 1 nM PEDF resulted in a complete inhibition of capillary alignment and tube formation in cultured GRECs, and also severely affected the formation of capillary-like tubes. Similar results were observed in assays carried out using a combination of EPO and PEDF. Fig. 3B shows the quantitative measurement of tube length. The tube formation assay shows that PEDF inhibits the EPO-induced tube formation in GRECs. 3.5. Involvement of PI3K/Akt in EPO-induced cell proliferation Phosphatidylinositol 3-kinase (PI3K) and its downstream regulator, Akt plays a critical role in the regulation of cellular processes, mainly proliferation and survival. To determine the involvement of PI3K/Akt in EPO-induced cell proliferation, LY294002, a specific inhibitor of PI3K pathway is used. GRECs were pre-treated with LY294002 (10 mM) for 15 min, and then treated with EPO (1 IU) for 24 h. The activity of PI3K/Akt in cell proliferation was determined by the MTT assay. It was found that pre-treatment with LY294002 inhibited endothelial cell survival induced by EPO and the inhibition was decreased by 3-fold as the proliferation seen with EPO alone (Fig. 4A). Further, to support the role of Akt in EPO-induced cell proliferation, GRECs were transfected with dominant negative Akt (DN Akt) and constitutively active Akt (CA Akt) mutant. Over expression of DN Akt blocked EPO-induced cell proliferation to the level of control, whereas over expression of CA Akt leads to increase in the endothelial cell proliferation. CA Akt rescues the inhibitory effect of PEDF in cell proliferation when compared the effects in
3.8. PEDF inhibits EPO-induced endothelial cell permeability We also examined the inhibitory effect of PEDF on EPO-induced endothelial cell permeability. In this experiment, PEDF was added 15 min prior to EPO treatment. PEDF inhibition of EPO-induced permeability occurred in a dose-dependent manner; 10 nM PEDF was sufficient to inhibit EPO-induced permeability to the level of control (Fig. 6). This result suggests that PEDF completely abrogated the EPO-induced increase in permeability to RITC-dextran. 3.9. Effect of PI3K/Akt inhibitor on EPO-induced endothelial cell permeability
We investigated the involvement of the PI3-kinase pathway in EPO-induced permeability in GRECs. Endothelial cells were grown to confluence, and solute flux was determined in the presence of EPO and LY294002. GRECs were pre-incubated with 10-mM LY294002 inhibitor for 15 min followed by the addition of 2 IU/ml of EPO and incubated for 24 h. It was found that pre-treatment with LY294002 inhibited endothelial cell permeability in the presence or absence of EPO treatment (Fig. 7A). This result suggests that EPOinduced cell permeability via PI3-kinase pathway in GRECs. 3.10. PEDF inhibits the EPO-induced permeability via PI3K/Akt pathway To verify further the role of PI3K/Akt activity in the regulation of vascular permeability, GRECs were transfected with plasmid encoding dominant negative (K179M) PI3K/Akt (DN Akt) and constitutive (myristoylated) Akt (CA Akt), grown to confluence and the effect of EPO and PEDF on endothelial cell permeability was measured. Over expression of DN Akt blocked EPO-induced
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Fig. 2. Effect of EPO on the migration of GRECs. In the presence of EPO, the GREC migrated around 90%, when compared to PEDF and controls and the difference was statistically significant.
permeability to the level of control, whereas over expression of CA Akt increased the permeability in GRECs. Stimulation of these cells with EPO treatment had a significant effect on cell permeability, which indicates that PI3K/Akt activation is sufficient for the induction of endothelial cell permeability (Fig. 7B). Further, CA Akt
rescues the inhibitory effect of PEDF-induced permeability when compared the effect of permeability in wild type GRECs. These data shows that EPO could induce the permeability via PI3K/Akt pathway, and PEDF inhibits the EPO-induced permeability via PI3K/Akt activity in GRECs.
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Fig. 3. Effect of EPO and PEDF on the differentiation (tube formation) of GRECs. In the control of 1% serum (A) and presence of EPO (B), the GRECs organized into a delicate capillary network. EPO caused an increase in tube length, when compared to both the kinds of PEDF-treated cells (C & D).
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4. Discussion
Fig. 4. (A) GRECs were treated with specific inhibitor for PI3K/Akt pathway. The inhibitor, LY294002, blocked the survival of the EPO-induced cells (0.037 0.006, P < 0.05, n ¼ 3). (B) GRECs were transfected on CA Akt and DN Akt; the cells were treated with EPO and PEDF. The CA Akt transfected cells treated with EPO given the more cell proliferation (SD: 2.87 0.0956667, P < 0.05, n ¼ 3), in that the DN Akt transfected cell treated with EPO significantly inhibit the cell proliferation (SD: 0.489 0.163, P < 0.05, n ¼ 3).
The present study demonstrates the anti-angiogenic and antipermeability effect of PEDF on EPO-induced proliferation and permeability in a retinal endothelial cell system from goat retina. In addition, we have demonstrated that PEDF inhibits EPO-induced proliferation and permeability by interrupting PI3K/Akt pathway. EPO is a pivotal mitogen that play a vital role in erythropoiesis and it also act as a potent ischemia-induced angiogenic factor (Yoshiya et al., 2005; Daisuke et al., 2005). Recent reports also suggest that EPO act as a survival factor for endothelial cells (Banumathi et al., 2009) and as a protects for the neuronal cells in the central nervous system and the retina. The level of EPO is significantly higher in patients with PDR, both in blood and vitreous humor (Daisuke et al., 2005) and treatment of EPO in diabetic mouse induces angiogenesis such as wound healing (Galeano et al., 2004). Moreover, it was reported that EPO induced the proliferation of ECs cultured in vitro (Rachel and Aly, 2007). In corroboration with all the above results, we have shown that EPO-induced the proliferation, migration and tube formation in GRECs. Moreover, vascular hyper-permeability occurs at the early stages of Diabetic Retinopathy, leading to neovascularization. Previously many angiogenic agents like VEGF, IL-1b have shown to induce permeability and cause neovascularization (Sheikpranbabu et al., 2009b). Here, EPO induced both cell proliferation and permeability in cultured GRECs. Moreover, consistent with the previous reports (Daisuke et al., 2005; Galeano et al., 2004), our results also show that EPO significantly induces the migration and tube formation. PEDF is a well-characterized anti-angiogenic molecule, which induces apoptosis in budding endothelial cells (Leiling et al., 2006). During the condition of diabetic retinopathy, there is a decrease in the level of PEDF in vitreous humor (Kayako et al., 2008) therefore further characterization of PEDF expression and its biological
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Fig. 5. Dose and time dependent effect of EPO on endothelial cell permeability. (A) GREC was grown to confluent monolayer on 12-well transwell insert and treated with various concentration of EPO, and RITC-dextran from the upper to the lower chamber was measured 6, 12, 18 and 24 h after treatment. Values are expressed in relative fluorescence counts (RFUs) as mean SEM, with each condition performed at least in triplicate (n ¼ 3, *P < 0.05). (B) GREC was grown to confluent monolayer on 12-well transwell insert and treated with various concentration of PEDF, and RITCdextran from the upper to the lower chamber was measured 6, 12, 18 and 24 h after treatment. Values are expressed in relative fluorescence counts (RFCs) as mean SEM, with each condition performed at least in triplicate (n ¼ 3, *P < 0.05).
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effects may offer new therapeutic approaches to prevent blindness. Thus, to confirm the role of PEDF as an anti-angiogenic molecule, we studied the effect of PEDF on EPO-induced proliferation and permeability in GRECs. PEDF significantly blocked the GRECs
Fig. 6. Effect of PEDF on EPO-induced endothelial cell permeability. GREC was grown to confluent monolayer on transwell insert membrane and incubated with EPO and/or PEDF, and the RITC-dextran from the upper to the lower chamber was measured 12 after treatment. PEDF was added 30 min prior to EPO treatments. Values are expressed in relative fluorescence counts (RFCs) as mean SEM, with each condition performed at least in triplicate.
Fig. 7. Role of PI3-kinase activity in EPO-induced endothelial cell permeability. (A) GREC was grown to confluent monolayer on transwell insert membranes. The lower chamber was incubated with EPO (2 IU/ml) in the presence or absence of LY294002 (10 mM) for 12 h at 37 C. LY294002 was added 30 min prior to growth factor treatments. The RITCdextran from the upper and lower chamber was measured 12 h after the treatment. (B) GREC was transiently transfected with DNA encoding GFP (pmaxGFP) or dominant negative Akt (K179M) or constitutive active Akt (myristoylated). Transfected GREC was grown to confluent monolayer and then incubated with EPO (2 IU/ml) for 12 h at 37 C. The RITC-dextran from the upper to the lower chamber was measured 12 h after the treatment. Values are expressed in relative fluorescence counts (RFCs) as mean SEM, with each condition performed at least in triplicate.
proliferation and permeability in the presence or absence of EPO. Further it also blocked the other steps in angiogenesis like cell migration and tube formation GRECs. Thus, PEDF blocked EPOinduced angiogenesis and permeability. Further, we studied the signaling mechanism using LY294002, a well-known inhibitor of PI3 Kinase. PI3K and its downstream target molecule serineethreonine kinase (Akt) are important for the regulation of a number of cellular responses (Rachel and Aly, 2007). LY294002 significantly inhibited the GRECs proliferation and permeability in the presence or absence of EPO as like as PEDF. In order to characterize the downstream targets of PI3-Kinase signaling, transfection assays were performed with DN Akt and CA Akt mutants which suggested that EPO-induced cell proliferation and permeability signals via PI3K/Akt activity in GRECs. Our data indicate that suppression of PI3-kinase expression would be the central mechanism for the anti-proliferative and anti-permeability effects of PEDF, thus providing a novel therapeutic potential of PEDF for the treatment of diabetic retinopathy. Although PEDF is still not approved as a therapeutic agent for angiogenic related disorders, various experiments have been performed on mice so as to confirm the action of PEDF. As mentioned previously, PEDF only act on budding endothelial cells in rat model.
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This work was supported in part by Department of Science and Technology (Grant No: SR/FT/L-118/2006), Department of Biotechnology (Grant No: BT/PR770/Med/14/1065/2006) and Indian Council of Medial Research (Grant No: 53/5/2006-BMS) Government of India, New Delhi. The author has gratefully acknowledged to Mr. V. Deepak for the critical reading of this manuscript.
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Acknowledgement
References
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Thromb. 34, 164e168. Mori, K., Gehlbach, P., Yamamoto, S., Duh, E., Zack, D.J., Li, Q., Berns, I.K., Raisler, J.B., Hauswirth, W.W., Campochiaro, A.P., 2002. AAV-mediated gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 43, 1994e2000. Nahoko, O., Maki, N., Tetsuya, N., Yumiko, M., Miyo, M., 2002. Inverse levels of pigment epithelium-derived factor and vascular endothelial growth factor in the vitreous of eyes with rhegmatogenous retinal detachment and proliferative vitreoretinopathy. Am. J. Ophthalmol. 133, 851e852. Olga, V.V., Tetiana, Z., Wei, Z., Frank, R., Thomas, A.F., Michael, S.P., Mohammad, A., Noel, P.B., 2002. Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium derived factor. Nat. Med. 8, 349e357. Rachel, Z., Aly, K., 2007. Erythropoietin promotes survival of primary human endothelial cells through PI3K-dependent, NF-kB-independent upregulation of Bcl-xL. Am. J. Physiol. Heart Circ. Physiol. 292, H2467eH2474. Raul, G.C., Alvaro, A.R., Marcos, R., 1995. Recombinant human erythropoietin stimulates angiogenesis in vitro. Kidney Int. 47, 740e745. Ray, G.F., Thomas, W.G., 2005. Retinal angiogenesis in development and disease. Nature 438, 960e966. Sarah, M., Jacobs, H., Kalyani, P., Zhenhong, S., Amy, L., Stephen, T.S., 1997. Distinct signaling from stem cell factor and erythropoietin in HCD57 cells. J. Biol. Chem. 272, 6850e6853. Sawant, S., Aparicio, S., Tink, A.R., Lara, N., Barnstable, C.J., Tombran, T.J., 2004. Regulation of factors controlling angiogenesis in liver development: a role for PEDF in the formation and maintenance of normal vasculature. Biochem. Biophys. Res. Commun. 325, 408e413. Semkova, I., Kreppel, F., Welsandt, G., Luther, T., Kozlowski, J., Janicki, H., Kochanek, S., Shraermeyer, U., 2002. Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye. Proc. Natl. Acad. Sci. 99, 13090e13095. Sheikpranbabu, S., Haribalaganesh, R., Banumathi, E., Sirishkumar, N., Jin Lee, K., Gurunathan, S., 2009a. Pigment epithelium-derived factor inhibits advance glycation end-product-induced angiogenesis and stimulates apoptosis in retinal endothelial cells. Life Sci. 85, 719e731. Sheikpranbabu, S., Kalishwaralal, K., Venkataraman, D., Eom, S.H., Park, J., Gurunathan, S., 2009b. Silver nanoparticles inhibit VEGF-and IL-1beta-induced vascular permeability via Src dependent pathway in porcine retinal endothelial cells. J. Nanobiotechnol. 7, 8. Steen, V.P., Zuzana, V., Jan, J.E., 2003. Pigment-epithelium-derived factor (PEDF) occurs at a physiologically relevant concentration in human blood: purification and characterization. Biochem. J. 374, 199e206. Tombran, T.J., Johnson, L.V., 1989. Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells. Invest. Ophthalmol. Vis. Sci. 30, 1700e1707. Veronica, S., Susan, E.C., Wei, Z., Noël, B., 2001. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epitheliumderived factor. Proc. Natl. Acad. Sci. 98, 2593e2597. Xi-yuan, Z., Qing, L., Yi-min, P., Yong-qiang, T., Xiao, G., Jia, L.I., Yan, X.U., Zhi-gang, W., 2009. Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization. Chin. Med. J. 122, 2711e2717. Yoshiya, K., Tadashi, O., Kazuhisa, M., Masahiro, O., Masahiko, K., Toshiya, W., Yoshiki, I., Masayuki, N., Hiroshi, K., Hideo, N., Takeshi, M., 2005. Erythropoietin is highly elevated in vitreous fluid of patients with proliferative diabetic retinopathy. Diabetes Care 28, 2252e2254.
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PEDF also induces differentiation in retinal neuroblastoma cells and also possesses neurotrophic function (Semkova et al., 2002). Moreover various procedures have been checked to increase the transfer of PEDF at the target site like lipofection, ultra sound (micro-bubble) and increasing the expression of the gene mediated by adenovirus (Xi-yuan et al., 2009). The adenovirus mediated transfer has reduced the angiogenesis in mouse model (Mori et al., 2002). Currently, PEDF has been transferred to human eye through adenovirus in Phase-1 clinical trial, which showed that the adenovirus to be safe and well tolerated by the patients and only 25% of patients showed the signs of inflammation suggesting adenoviral mediated PEDF treatment to be safe for ocular therapy (Campochiaro et al., 2006). In summary, this report demonstrates that PEDF inhibits EPOinduced proliferation, migration, tube formation and permeability in GRECs. Furthermore, PEDF significantly suppress EPO-induced PI3K/Akt signaling in endothelial cells. To our knowledge, this is the first study that demonstrates the anti-proliferative and antipermeability effects of PEDF on cultured endothelial cells. The effect of PEDF on other cell survival pathways induced by EPO in retinal endothelial cells need to be further investigated.
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Banumathi, E., Haribalaganesh, R., Sheik Pran Babu, S., Kyung, J.L., Sangiliyandi, G., 2009. PEDF inhibits VEGF- and EPO-induced angiogenesis in retinal endothelial cells through interruption of PI3K/Akt phosphorylation. Angiogenesis 12, 313e324. Bilak, M.M., Corse, A.M., Bilak, S.R., Lehar, M., Tombran, T.J., Kuncl, R.W., 1999. Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration. J. Neuropathol. Exp. Neurol. 58, 719e728. Böcker-Meffert, S., Rosenstiel, P., Röhl, C., Warneke, N., Held-Feindt, J., Sievers, J., Lucius, R., 2002. Erythropoietin and VEGF promote neural outgrowth from retinal explants in postnatal rats. Invest. Ophthalmol. Vis. Sci. 43, 2021e2026. Campochiaro, A.P., Nguyen, D.Q., Shah, S.M., Klein, L.M., Holz, E., Frank, N.R., Saperstein, A.D., Gupta, A., Stout, T.J., Macko, J., Dibartolomeo, R., Wei, L.L., 2006. Adenoviral vector-delivered pigment epithelium-derived factor for neovascular age-related macular degeneration: results of a phase I clinical trial. Hum. Gene Ther. 17, 167e176. Daisuke, W., Kiyoshi, S., Shigeyuki, M., Masafumi, K., Junichi, K., Mihori, K., Izumi, S., Hirokazu, O., Tomonari, O., Tomoaki, M., Toshihiro, K., Seiji, M., Masaya, N., Nagahisa, Y., Hitoshi, T., 2005. Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy. N. Engl. J. Med. 353, 782e792. Dawson, D.W., Volpert, O.V., Gillis, P., Crawford, S.E., Xu, H.J., Benedict, W., Bouck, N.P., 1999. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science 285, 245e248. Domenico, R., Marco, P., Angelo, V., Roberto, R., Roberta, G., Patrizia, D., Beatrice, N., Luisa, R., Franco, D., 1999. Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo. Blood 93, 2627e2636. Dolores, L.M., Maaza, G.A., Dilip, K.D., Leonidas, C.P., Shahab, U., Amittha, W., 2002. Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells. Oncogene 21, 1556e1562. Frank, R.N., 1991. On the pathogenesis of diabetic retinopathy. Ophthalmology 98, 586e593. Galeano, M., Altavilla, D., Cucinotta, D., Russo, G.T., Calo, M., Bitto, A., Marini, H., Marini, R., Adamo, B.E., Seminara, P., Minutoli, L., Torre, V., Squadrito, F., 2004. Recombinant human erythropoietin stimulates angiogenesis and wound healing in the genetically diabetic mouse. Diabetes 53, 2509e2517. Gerald, M.Y., Quan, J.O., Yaping, L., Vicky, K., Hong, Z., Peter, F.M.C., 2005. Localization of pigment epithelium-derived factor in growing mouse bone. Calcif. Tissue Int. 76, 146e153. Haribalaganesh, R., Sheikpranbabu, S., Elayappan, B., Venkataraman, D., Gurunathan, S., 2009. Pigment epithelium-derived factor down regulates
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Pigment epithelium-derived factor inhibits erythropoietin-induced retinal endothelial cell angiogenesis by suppression of PI3K/Akt pathway Ravinarayanan Haribalaganesh, Sardarpasha Sheikpranbabu, Elayappan Banumathi, Sangiliyandi Gurunathan*
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Department of Biotechnology, Division of Molecular and Cellular biology, Kalasalingam University (Kalasalingam Academy of Research and Education), Anand Nagar, Krishnankoil 626190, Tamilnadu, India
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Article history: Received 27 September 2009 Accepted in revised form 10 March 2010 Available online 16 March 2010
Erythropoietin (EPO) plays a critical role in the vascular system and exhibits angiogenic activity in endothelial cells (ECs) such as stimulation of cell proliferation, migration and tube formation in vitro. EPO is the major regulator of cell proliferation and differentiation of erythroid precursors and there by preventing the apoptosis. Pigment epithelial derived factor (PEDF) is a potent anti-angiogenic factor whose effects are partially mediated through the induction of EC apoptosis. The mechanism of EPO and PEDF in retinal neovascularization has not been well documented yet. The effect of EPO and PEDF on cell proliferation was determined by MTT assay. In vitro wound-scratch assay was performed to study the migration of ECs and in vitro tube formation was assessed by the on-gel assay system using gelatin. Inhibitor assay was carried out using LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Further, PI3K/Akt activity was assessed by transient transfection assay using constitutively active (CA) and dominant negative (DN) Akt mutants. Dextran permeability assay was performed to determine the vascular permeability. We report that EPO stimulates EC proliferation, migration, tube formation and permeability whereas PEDF inhibits the EPO-induced ECs proliferation and permeability. Over expression of DN Akt blocked EPO stimulation of proliferation and permeability, while over expression of CA Akt rescues the inhibitory effect of PEDF on proliferation and permeability. These results demonstrate that PEDF may inhibit the EPO-induced proliferation and permeability via PI3K/Akt-dependent pathway. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
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Keywords: endothelial cells erythropoietin pigment epithelium-derived factor proliferation vascular permeability
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a r t i c l e i n f o
Angiogenesis is thought to depend on a precise balance of positive and negative regulation. The neovascularization of the retina occurs predominantly via angiogenesis, which is the formation of functional blood vessels from a pre-existing vasculature (Ray and Thomas, 2005). Although this process was tightly regulated under physiological conditions, persistent unregulated angiogenesis is a hallmark of diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity and age-related macular degeneration. Diabetic retinopathy is one of the significant microvascular complications in diabetes and is a leading cause of acquired blindness among people of occupational age (Frank, 1991). The vascular permeability and its regulatory control are central to homeostasis and increases in vascular permeability play a key role in the development of sepsis-associated hypotension, acute
* Corresponding author. Tel.: þ91 4563 289042; fax: þ91 4563 289322. E-mail address: [email protected] (S. Gurunathan). 0014-4835/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2010.03.005
respiratory distress syndrome, nephrotic syndrome, diabetic retinopathy and diabetic neuropathy. Erythropoietin (EPO) is a 34-kDa renal glycoprotein hormone, responsible for the development of immature erythroid cells (Koury and Bondurant, 1992; Sarah et al., 1997) and was first characterized as a hematopoietic factor. More recently, EPO and EPO receptor (EPOR) were found to be expressed by brain and retinal tissue (Jelkmann and Metzen, 1996; Hugo et al., 2006; Juul et al., 1998; Böcker-Meffert et al., 2002). Besides an increase of intracellular calcium, the main signaling pathways of hematopoietic cells involved in the activation of phosphatidylinositol 3-kinase (PI3K/Akt), MAPK, JAK and activation of transcription factor 5 (STAT5) for survival, proliferation, and migration of stimulated cells (Raul et al., 1995; Domenico et al., 1999; Dolores et al., 2002). Pigment epithelium-derived factor (PEDF) is a 50-kDa secreted glycoprotein that was first isolated from conditioned medium of cultured primary human fetal retinal pigment epithelial cells (Tombran and Johnson, 1989). PEDF is widely expressed throughout fetal and adult tissues, including the adult brain (Bilak et al., 1999), spinal cord (Kuncl et al., 2002), plasma (Steen et al., 2003),
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2. Materials and methods 2.1. Cell culture
GRECs were seeded at a density of 2 103 cells per well into 96well culture plates. After attachment, the culture medium was changed to IMDM containing 10% FBS, and the cells were grown for 24 h. After reaching 80% confluence, the cells were starved with IMDM containing 1% FBS for 5 h and then treated with various concentrations of EPO (Sigma St Louis, USA) or PEDF (Abcam, Cambridge, UK) and/or combinations of both. After 24 h, cell proliferation was assessed using the MTT assay as per the manufacturer’s instructions (MTT-cell proliferation kit, Roche, Germany GmbH). Absorbance was measured at 595 nm using an enzymelinked immunosorbent assay (ELISA) plate reader (Biorad Model 680, Japan). 2.3. In vitro cell migration assay
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To examine the cell migration effect of EPO and PEDF on GRECs, a wound-scratch assay was followed as described previously (Mary et al., 2005). Cells were seeded in 35-mm plates and grown to confluence in IMDM growth medium. The monolayer was starved for 5 h in IMDM with 1% FBS. Cells were treated with 1 mM 5-Fluorouracil (Sigma St Louis, MO) for 15 min to rule out the contribution of differences in cell proliferation. Endothelial cells were aseptically scraped out from the middle of the plate using a sterile 200-ml micropipette tip rinsed with growth medium. After scraping, cells were rinsed twice with medium to remove woundderived cells. Then the cells were scratched with a plastic pipette tip to yield cell-free areas of uniform width and treated with EPO and PEDF. Control plates contained pure assay medium (1% serum, without growth factors). After incubation at 37 C for time intervals of 0 and 24 h, movement of cells into the denuded area was evaluated. To determine the extent of wound closure, digital photographs were taken (two pictures from each plate at randomly chosen areas) after 24 h and the wound closer area was analyzed by axiovision software (Zeiss, Germany).
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liver (Sawant et al., 2004), bone (Gerald et al., 2005), eye, heart, and lung (Joyce et al., 1996). It is one of the most potent endogenous inhibitor of angiogenesis in the eye (Dawson et al., 1999; Veronica et al., 2001). Several studies have shown that a decreased level of PEDF in the eye is associated with a number of ocular neovascular and neurodegenerative diseases. Inhibition of vessel formation was mediated by up-regulation of the Fas-ligand on the surface of endothelial cells, with subsequent induction of apoptosis in actively dividing cells (Olga et al., 2002). PEDF selectively inhibits the formation of new vessels from endothelial cells but does not appear to harm the existing vascular structure. The inhibitory effect of PEDF on vessel formation appears to be reversible when a transient and regulated angiogenesis occurs in situations including tissue repair after injury. A recent study has provided clinical evidence to show that expression of VEGF and PEDF is inversely correlated during the development of retinal neovascularization, further supporting an anti-angiogenic role for PEDF in-vivo (Nahoko et al., 2002). Furthermore the high expression of EPO and low expression of PEDF has been established in angiogenesis but the correlation effect of the EPO and PEDF remains unclear. In the present study, we have investigated the molecular mechanism of PEDF on EPO-induced angiogenesis, and permeability in goat retinal microvascular endothelial cells (GRECs). We report that EPO increases the cell proliferation, migration, tube formation and permeability significantly in GRECs. Further, PEDF inhibits the EPOinduced via angiogenesis and permeability via PI3K/Akt-dependent pathway and this pathway might represent a potential target to inhibit the neovascularization process, which may lead to the development of novel anti-neovascular agents for therapeutic purposes.
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Goat retinal microvascular endothelial cells (GRECs) were isolated and cultured as described previously (Sheikpranbabu et al., 2009a). Briefly, freshly isolated goat retinas were washed twice with sterile 1 Phosphate Buffer Saline (PBS), pH 7.2 and the retinal layer was separated in to retinal tissues and blood vessels. The blood vessels were minced and mixed with 1 PBS, transferred to a 15 ml tube, centrifuged at 600 g for 5 min at room temperature and the supernatant discarded. The pellet containing the blood vessels was suspended in 3 ml of proteolytic enzyme cocktail containing 0.01 g collagenase type IV, 0.004 g Pronase and 0.004 g DNAse, mixed with 2 ml of 1 PBS (pH 7.2), and incubated at 37 C for 20e25 min. Subsequently the digested blood vessels were centrifuged at 600 g for 5 min at room temperature and the supernatant discarded. The pellet containing the blood vessels was resuspended in 1.5 ml of fresh Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal bovine serum (FBS) (Sera Laboratories International Ltd, UK), 100 U/ml Penicillin-G, 100 mg/ml Streptomycin and 250 mg/ml Amphotericin-B. The suspended blood vessels were seeded in 35-mm tissue culture dish pre-coated with 1% gelatin, incubated at 37 C in 5% CO2 atmosphere. After 12 h the unattached blood vessels and debris were removed and washed once with serum free IMDM followed by the addition of fresh medium supplemented with 10% FBS and the cultures returned to 37 C with 5% CO2. The medium was changed every 3 days and the cell growth was monitored under phase contrast microscope. The cells were used between passages 4 and 9. 2.2. In vitro cell proliferation assay To assess the cell proliferation, a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay was used. Briefly,
2.4. In vitro capillary morphogenesis assay
The isolated GRECs were seeded at 3 104 cells in 35-mm tissue culture plates pre-coated with 4% gelatin. After 24 h, the growth medium was replaced with IMDM medium containing 0.5% FBS and PEDF in the presence or absence of EPO in GRECs and the plates were maintained at 37 C with 5% CO2. The capillary tube formation was observed under phase contrast microscope (Zeiss Axiovert, Germany) and the tubular length was measured using axiovision software. 2.5. Pharmacological inhibitor assay GRECs were seeded at 2 103 cells per well in a 96-well plate pre-coated with 1% gelatin. After 24 h, the growth medium was replaced with IMDM containing 0.5% FBS. The cells were treated with PI3K specific pharmacological inhibitor, LY294002 (10 mM) for 15 min prior to EPO treatment. The cell proliferation rate was analyzed using MTT assay according to the manufacturer’s instructions and the cell proliferation was measured using a micro plate Reader (Biorad Model 680, Japan). 2.6. Transient transfection assay Constitutively active (myristoylated) Akt (CA Akt) and dominant negative (K179M) Akt (DN Akt) mutants were kindly provided by Dr. Kyung-Jin Lee (Department of Life Science, GIST, Korea). Transient transfection assay was carried out using the method described earlier (Haribalaganesh et al., 2009). Briefly, GRECs were transiently transfected using a nucleofection technique
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(Amaxa Biosystems, Koeln, Germany) and grown to 80% confluence in IMDM and the cells were harvested by trypsinization and centrifuged at 3000 rpm for 10 min. The pellet was resuspended in the nucleofector solution (Basic nucleofector kit, Amaxa Inc., Germany) to a final concentration of 4e5 105 cells/100 ml. At the time of transfection, 1e3 mg of plasmid DNA encoding green fluorescent protein (pmaxGFP), CA Akt or DN Akt was added along with a nucleofector solution and subjected to electroporation using a nucleofector device-II (Amaxa Biosystems, Koeln, Germany). After electroporation, transfected cells were resuspended in 35-mm plates containing 1 ml of prewarmed IMDM and incubated in 5% CO2 at 37 C. The transfection efficiency determined after cotransfection with a pmaxGFP plasmid (Amaxa Inc.) and cell viability determined by MTT assay were usually about 80e90%.
3. Results 3.1. EPO induces the cell proliferation in GRECs Recent reports suggest that erythropoietin is a potent angiogenic molecule which induces cell survival and proliferation in retinal endothelial cells (Rachel and Aly, 2007). To determine the effect of EPO on cell proliferation in GRECs, cells were exposed to various concentration of EPO for 24 h. The effect of EPO on endothelial cell proliferation was determined by MTT colorimetric assay. EPO-induced maximal cell proliferation at 1 IU compared to control (Fig. 1A). This mitogenic effect was gradually lost as the concentration of EPO was increased in the medium to a maximum of 10 IU. The data suggest that EPO-induced cell proliferation significantly in GRECs.
2.7. Culture in permeable insert systems
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Endothelial cell permeability of a confluent cell monolayer was determined by a fluorescence-based assay as reported previously (Sheikpranbabu et al., 2009b). Briefly confluent monolayer endothelial cells (ECs) were incubated with IMDM assay medium for 5 h containing only 1% FBS without any growth factors. Here, we used Rhodamine isothiocyanate (RITC)-dextran (Sigma St Louis, MO) with a molecular weight of 70-kDa, which corresponds to a diameter of approximately 6 nm. The ability of the dextran to pass from the upper chamber of the permeable insert system into the lower chamber was used to determine the leakage properties of the monolayer. More dextran in the lower chamber indicates higher permeability. This dextran flux was quantified by means of the fluorescence in the lower chamber. 10 mM of RITC was applied to the apical chamber of the transwell inserts with a confluent ECs monolayer. Permeability assays started 4 days after seeding, when a confluent monolayer of ECs was present. EPO was added for the designated times. Where applicable, PEDF was added 15 min prior to EPO treatment. In some experiments, LY294002, a PI3-kinase inhibitor was added to ECs cultures 15 min prior to growth factor addition. The media volumes used equalized fluid heights in the apical and basolateral chambers, so that only diffusive forces were involved in solute permeability. At the indicated times after treatment, 100 ml samples were taken from the basolateral chamber and placed in a 96-well plate (96-well, polystyrene, flat bottom, Corning, USA). A sample was taken from the apical chamber at the last time point; the amount of fluorescence in this chamber did not change significantly over the course of the experiment. Aliquots were quantified using a fluorescence multi-detection micro plate reader (Biotek FLx800, Vermont, USA) at an excitation/emission of 540/590 nm.
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2.8. Dextran endothelial cell permeability assay
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GRECs at passage five were seeded in IMDM coated with gelatin in porous membranes of permeable insert system (Transwell, Corning, USA, pore size 0.4 mm, growth area 0.33 cm2) with a seeding density of 30,000 cells/cm2. The medium was changed the next day, and every other day thereafter. 500-ml medium was also added in the lower chamber of the insert systems.
2.9. Statistical analysis All results are expressed as the means standard deviation (SD) values. The statistical significance was evaluated by using the paired two-tailed student’s t test. A P < 0.05 was considered to indicate a statistically significant difference.
Fig. 1. (A) Effect of EPO on GRECs survival: GRECs were treated with different concentrations of EPO. Where 1 IU EPO induce Maximum proliferation (0.114 0.010, P < 0.05, n ¼ 3), (b) Effect of PEDF on GRECs survival: GRECs were treated with different concentrations of PEDF. Where 1 nM PEDF sufficiently inhibited the cell survival (0.037 0.006, P < 0.05, n ¼ 3), (C) Effect of PEDF on EPO-induced GRECs survival: 1 IU EPO-induced GRECs were treated with 1 nM PEDF. Where 1 nM PEDF inhibited the cell survival (0.063 0.030, P < 0.05, n ¼ 3).
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3.3. PEDF blocks EPO-induced endothelial cell migration
To study the ability of EPO to induce EC permeability, GRECs were grown to confluence on transwell membrane filters and then treated with EPO at various concentrations. EPO-induced dosedependent increase in permeability of the endothelial cell monolayer which is shown by the relative transfer of RITC-labeled dextran in 12 h, where maximal permeability was induced by the treatment with 2 IU/ml of EPO (Fig. 5A). The effect of EPO on permeability persisted over 24 h duration of the experiment. 3.7. PEDF inhibits endothelial cell permeability in a dose-dependent manner PEDF is known to act on multiple cell types in addition to endothelial cells such as neurons, glial cells, microglia, and retinal pigment epithelium, but we were interested in examining whether PEDF has a direct effect on endothelial cells in the regulation of permeability. To determine the role of PEDF on endothelial cell permeability, we examined the diffusion of non-radioactively labeled dextran across a confluent endothelial cell monolayer. PEDF reduced the permeability to RITC-dextran compared with control significantly (Fig. 5B). The inhibitory effect was dosedependent, reaching a maximum at 10 nM and a very less significant degree of inhibition occurred at lower doses. In addition, the effect of PEDF on permeability persisted over the 24 h duration of the experiment.
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Angiogenesis involves a variety of parallel events, including matrix degradation, migration, proliferation and morphogenesis of endothelial cells. To know the effect of PEDF and EPO on cell migration, a wound-scratch assay was performed. The assay showed that EPO significantly increases migration of endothelial cells; PEDF was observed to inhibit the cell migration. Fig. 2A shows enhanced migration of GRECs and wound closure being completed almost by 24 h in 1 IU EPO-treated plates, whereas a significant area of the wound remaining uncovered in 1 nM PEDF-treated plates. The same inhibitory effect was observed in plates incubated with combinations of EPO and PEDF, which shows that PEDF blocks the EPO-induced cell migration. Fig. 2B shows the quantitative assessments of cell migration under various conditions.
3.6. EPO enhances endothelial cell permeability in GRECs
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PEDF is a well-known inhibitor of angiogenesis and it is involved in EPO-induced barrier dysfunction of microvascular endothelial cells. To confirm the effect of PEDF on GRECs, cells were exposed to various concentrations of PEDF and incubated for 24 h. Among various concentrations of PEDF tested, 1 nM PEDF significantly inhibits the cell proliferation in GRECs (Fig. 1B). Further, to analyze the counteractive effect of PEDF on EPO-induced cell proliferation, 1 nM of PEDF was added 15 min prior to EPO treatment. The result shows that pre-treatment of PEDF significantly blocked the EPOinduced cell proliferation in GRECs (Fig. 1C).
wild type GRECs (Fig. 4B). These results show that EPO could possibly induce the cell survival via PI3K/Akt-dependent pathway, and PEDF might suppress the EPO-induced PI3K/Akt activity in GRECs.
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3.2. PEDF inhibits the EPO-induced cell proliferation
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3.4. PEDF inhibits EPO-induced tube formation in GRECs
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The ability of endothelial cells to form capillary-like structures on an extracellular matrix can distinguish endothelial cells from other common contaminating cell types. To further characterize the effect of EPO and PEDF on angiogenesis, the effect of EPO and PEDF on endothelial tube formation was analyzed using capillary morphogenesis assay. Fig. 3A shows that GRECs treated with 1 IU EPO organize well on the 4% gelatin and form extensive capillary networks. In contrast, the addition of 1 nM PEDF resulted in a complete inhibition of capillary alignment and tube formation in cultured GRECs, and also severely affected the formation of capillary-like tubes. Similar results were observed in assays carried out using a combination of EPO and PEDF. Fig. 3B shows the quantitative measurement of tube length. The tube formation assay shows that PEDF inhibits the EPO-induced tube formation in GRECs. 3.5. Involvement of PI3K/Akt in EPO-induced cell proliferation Phosphatidylinositol 3-kinase (PI3K) and its downstream regulator, Akt plays a critical role in the regulation of cellular processes, mainly proliferation and survival. To determine the involvement of PI3K/Akt in EPO-induced cell proliferation, LY294002, a specific inhibitor of PI3K pathway is used. GRECs were pre-treated with LY294002 (10 mM) for 15 min, and then treated with EPO (1 IU) for 24 h. The activity of PI3K/Akt in cell proliferation was determined by the MTT assay. It was found that pre-treatment with LY294002 inhibited endothelial cell survival induced by EPO and the inhibition was decreased by 3-fold as the proliferation seen with EPO alone (Fig. 4A). Further, to support the role of Akt in EPO-induced cell proliferation, GRECs were transfected with dominant negative Akt (DN Akt) and constitutively active Akt (CA Akt) mutant. Over expression of DN Akt blocked EPO-induced cell proliferation to the level of control, whereas over expression of CA Akt leads to increase in the endothelial cell proliferation. CA Akt rescues the inhibitory effect of PEDF in cell proliferation when compared the effects in
3.8. PEDF inhibits EPO-induced endothelial cell permeability We also examined the inhibitory effect of PEDF on EPO-induced endothelial cell permeability. In this experiment, PEDF was added 15 min prior to EPO treatment. PEDF inhibition of EPO-induced permeability occurred in a dose-dependent manner; 10 nM PEDF was sufficient to inhibit EPO-induced permeability to the level of control (Fig. 6). This result suggests that PEDF completely abrogated the EPO-induced increase in permeability to RITC-dextran. 3.9. Effect of PI3K/Akt inhibitor on EPO-induced endothelial cell permeability
We investigated the involvement of the PI3-kinase pathway in EPO-induced permeability in GRECs. Endothelial cells were grown to confluence, and solute flux was determined in the presence of EPO and LY294002. GRECs were pre-incubated with 10-mM LY294002 inhibitor for 15 min followed by the addition of 2 IU/ml of EPO and incubated for 24 h. It was found that pre-treatment with LY294002 inhibited endothelial cell permeability in the presence or absence of EPO treatment (Fig. 7A). This result suggests that EPOinduced cell permeability via PI3-kinase pathway in GRECs. 3.10. PEDF inhibits the EPO-induced permeability via PI3K/Akt pathway To verify further the role of PI3K/Akt activity in the regulation of vascular permeability, GRECs were transfected with plasmid encoding dominant negative (K179M) PI3K/Akt (DN Akt) and constitutive (myristoylated) Akt (CA Akt), grown to confluence and the effect of EPO and PEDF on endothelial cell permeability was measured. Over expression of DN Akt blocked EPO-induced
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Fig. 2. Effect of EPO on the migration of GRECs. In the presence of EPO, the GREC migrated around 90%, when compared to PEDF and controls and the difference was statistically significant.
permeability to the level of control, whereas over expression of CA Akt increased the permeability in GRECs. Stimulation of these cells with EPO treatment had a significant effect on cell permeability, which indicates that PI3K/Akt activation is sufficient for the induction of endothelial cell permeability (Fig. 7B). Further, CA Akt
rescues the inhibitory effect of PEDF-induced permeability when compared the effect of permeability in wild type GRECs. These data shows that EPO could induce the permeability via PI3K/Akt pathway, and PEDF inhibits the EPO-induced permeability via PI3K/Akt activity in GRECs.
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Fig. 3. Effect of EPO and PEDF on the differentiation (tube formation) of GRECs. In the control of 1% serum (A) and presence of EPO (B), the GRECs organized into a delicate capillary network. EPO caused an increase in tube length, when compared to both the kinds of PEDF-treated cells (C & D).
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4. Discussion
Fig. 4. (A) GRECs were treated with specific inhibitor for PI3K/Akt pathway. The inhibitor, LY294002, blocked the survival of the EPO-induced cells (0.037 0.006, P < 0.05, n ¼ 3). (B) GRECs were transfected on CA Akt and DN Akt; the cells were treated with EPO and PEDF. The CA Akt transfected cells treated with EPO given the more cell proliferation (SD: 2.87 0.0956667, P < 0.05, n ¼ 3), in that the DN Akt transfected cell treated with EPO significantly inhibit the cell proliferation (SD: 0.489 0.163, P < 0.05, n ¼ 3).
The present study demonstrates the anti-angiogenic and antipermeability effect of PEDF on EPO-induced proliferation and permeability in a retinal endothelial cell system from goat retina. In addition, we have demonstrated that PEDF inhibits EPO-induced proliferation and permeability by interrupting PI3K/Akt pathway. EPO is a pivotal mitogen that play a vital role in erythropoiesis and it also act as a potent ischemia-induced angiogenic factor (Yoshiya et al., 2005; Daisuke et al., 2005). Recent reports also suggest that EPO act as a survival factor for endothelial cells (Banumathi et al., 2009) and as a protects for the neuronal cells in the central nervous system and the retina. The level of EPO is significantly higher in patients with PDR, both in blood and vitreous humor (Daisuke et al., 2005) and treatment of EPO in diabetic mouse induces angiogenesis such as wound healing (Galeano et al., 2004). Moreover, it was reported that EPO induced the proliferation of ECs cultured in vitro (Rachel and Aly, 2007). In corroboration with all the above results, we have shown that EPO-induced the proliferation, migration and tube formation in GRECs. Moreover, vascular hyper-permeability occurs at the early stages of Diabetic Retinopathy, leading to neovascularization. Previously many angiogenic agents like VEGF, IL-1b have shown to induce permeability and cause neovascularization (Sheikpranbabu et al., 2009b). Here, EPO induced both cell proliferation and permeability in cultured GRECs. Moreover, consistent with the previous reports (Daisuke et al., 2005; Galeano et al., 2004), our results also show that EPO significantly induces the migration and tube formation. PEDF is a well-characterized anti-angiogenic molecule, which induces apoptosis in budding endothelial cells (Leiling et al., 2006). During the condition of diabetic retinopathy, there is a decrease in the level of PEDF in vitreous humor (Kayako et al., 2008) therefore further characterization of PEDF expression and its biological
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Fig. 5. Dose and time dependent effect of EPO on endothelial cell permeability. (A) GREC was grown to confluent monolayer on 12-well transwell insert and treated with various concentration of EPO, and RITC-dextran from the upper to the lower chamber was measured 6, 12, 18 and 24 h after treatment. Values are expressed in relative fluorescence counts (RFUs) as mean SEM, with each condition performed at least in triplicate (n ¼ 3, *P < 0.05). (B) GREC was grown to confluent monolayer on 12-well transwell insert and treated with various concentration of PEDF, and RITCdextran from the upper to the lower chamber was measured 6, 12, 18 and 24 h after treatment. Values are expressed in relative fluorescence counts (RFCs) as mean SEM, with each condition performed at least in triplicate (n ¼ 3, *P < 0.05).
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effects may offer new therapeutic approaches to prevent blindness. Thus, to confirm the role of PEDF as an anti-angiogenic molecule, we studied the effect of PEDF on EPO-induced proliferation and permeability in GRECs. PEDF significantly blocked the GRECs
Fig. 6. Effect of PEDF on EPO-induced endothelial cell permeability. GREC was grown to confluent monolayer on transwell insert membrane and incubated with EPO and/or PEDF, and the RITC-dextran from the upper to the lower chamber was measured 12 after treatment. PEDF was added 30 min prior to EPO treatments. Values are expressed in relative fluorescence counts (RFCs) as mean SEM, with each condition performed at least in triplicate.
Fig. 7. Role of PI3-kinase activity in EPO-induced endothelial cell permeability. (A) GREC was grown to confluent monolayer on transwell insert membranes. The lower chamber was incubated with EPO (2 IU/ml) in the presence or absence of LY294002 (10 mM) for 12 h at 37 C. LY294002 was added 30 min prior to growth factor treatments. The RITCdextran from the upper and lower chamber was measured 12 h after the treatment. (B) GREC was transiently transfected with DNA encoding GFP (pmaxGFP) or dominant negative Akt (K179M) or constitutive active Akt (myristoylated). Transfected GREC was grown to confluent monolayer and then incubated with EPO (2 IU/ml) for 12 h at 37 C. The RITC-dextran from the upper to the lower chamber was measured 12 h after the treatment. Values are expressed in relative fluorescence counts (RFCs) as mean SEM, with each condition performed at least in triplicate.
proliferation and permeability in the presence or absence of EPO. Further it also blocked the other steps in angiogenesis like cell migration and tube formation GRECs. Thus, PEDF blocked EPOinduced angiogenesis and permeability. Further, we studied the signaling mechanism using LY294002, a well-known inhibitor of PI3 Kinase. PI3K and its downstream target molecule serineethreonine kinase (Akt) are important for the regulation of a number of cellular responses (Rachel and Aly, 2007). LY294002 significantly inhibited the GRECs proliferation and permeability in the presence or absence of EPO as like as PEDF. In order to characterize the downstream targets of PI3-Kinase signaling, transfection assays were performed with DN Akt and CA Akt mutants which suggested that EPO-induced cell proliferation and permeability signals via PI3K/Akt activity in GRECs. Our data indicate that suppression of PI3-kinase expression would be the central mechanism for the anti-proliferative and anti-permeability effects of PEDF, thus providing a novel therapeutic potential of PEDF for the treatment of diabetic retinopathy. Although PEDF is still not approved as a therapeutic agent for angiogenic related disorders, various experiments have been performed on mice so as to confirm the action of PEDF. As mentioned previously, PEDF only act on budding endothelial cells in rat model.
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This work was supported in part by Department of Science and Technology (Grant No: SR/FT/L-118/2006), Department of Biotechnology (Grant No: BT/PR770/Med/14/1065/2006) and Indian Council of Medial Research (Grant No: 53/5/2006-BMS) Government of India, New Delhi. The author has gratefully acknowledged to Mr. V. Deepak for the critical reading of this manuscript.
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Acknowledgement
References
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PEDF also induces differentiation in retinal neuroblastoma cells and also possesses neurotrophic function (Semkova et al., 2002). Moreover various procedures have been checked to increase the transfer of PEDF at the target site like lipofection, ultra sound (micro-bubble) and increasing the expression of the gene mediated by adenovirus (Xi-yuan et al., 2009). The adenovirus mediated transfer has reduced the angiogenesis in mouse model (Mori et al., 2002). Currently, PEDF has been transferred to human eye through adenovirus in Phase-1 clinical trial, which showed that the adenovirus to be safe and well tolerated by the patients and only 25% of patients showed the signs of inflammation suggesting adenoviral mediated PEDF treatment to be safe for ocular therapy (Campochiaro et al., 2006). In summary, this report demonstrates that PEDF inhibits EPOinduced proliferation, migration, tube formation and permeability in GRECs. Furthermore, PEDF significantly suppress EPO-induced PI3K/Akt signaling in endothelial cells. To our knowledge, this is the first study that demonstrates the anti-proliferative and antipermeability effects of PEDF on cultured endothelial cells. The effect of PEDF on other cell survival pathways induced by EPO in retinal endothelial cells need to be further investigated.
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