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Activation of Vascular Endothelial Growth Factor Receptor-1 Sustains Angiogenesis and Bcl-2 Expression Via the Phosphatidylinositol 3-Kinase Pathway in Endothelial Cells Jun Cai,1 Shakil Ahmad,2 Wen G. Jiang,3 Jianhua Huang,4 Christopher D. Kontos,4 Mike Boulton,1 and Asif Ahmed2

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide–stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation From the 1Department of Optometry and Vision Sciences, Cardiff University, Cardiff, U.K.; the 2Department of Reproductive and Vascular Biology, Medical School, University of Birmingham, Birmingham, U.K.; the 3Department of Surgery, University of Wales College of Medicine, Cardiff, U.K.; and the 4 Department of Medicine, Duke University Medical Center, Durham, North Carolina. Address correspondence and reprint requests to Professor Asif Ahmed, Department of Reproductive and Vascular Biology, University of Birmingham, the Medical School, Edgbaston, Birmingham B15 2TG, U.K. E-mail: [email protected] Received for publication 19 February 2003 and accepted in revised form 27 August 2003. J.C. and S.A. have made equal contributions to this study. AdEV, empty virus; AdPTEN-C/S, adenovirus of catalytic inactive mutant of phosphatase and tension homolog on chromosome 10; AdPTEN-WT, adenovirus of wild-type phosphatase and tensin homolog on chromosome 10; BREC, bovine retinal endothelial cell; MECBM, microvascular endothelial cell basal medium; PDGF, platelet-derived growth factor; PlGF, placenta growth factor; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog on chromosome 10; TNF-␣, tumor necrosis factor-␣; VEGF, vascular endothelial growth factor, VEGFR, VEGF receptor. © 2003 by the American Diabetes Association. DIABETES, VOL. 52, DECEMBER 2003

and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks. Diabetes 52:2959 –2968, 2003

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ascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of vascular complications in diabetes (1). VEGF is secreted by numerous ocular cell types (1), and increased levels of VEGF have been detected in ocular fluids of patients with proliferative diabetic retinopathy (2). In vivo, administration of neutralizing VEGF antibodies to experimental animals reverses high-glucose–induced vascular hyperpermeability (3), which is an early manifestation of endothelial dysfunction in diabetic patients (4). Placenta growth factor (PIGF), a secreted dimeric protein of the VEGF family, shares a 53% sequence identity with the platelet-derived growth factor (PDGF)-like region of VEGF (5). Similarly to VEGF, alternative splicing of PlGF mRNA produces at least three polypeptides of 149 (PlGF-1), 170 (PlGF-2), and 221 (PlGF-3) amino acids (6,7). A highly basic 21–amino acid insertion in the COOHterminal region of PlGF-2 sequence results in the high heparin-binding affinity of this polypeptide. PlGF binds only to VEGF receptor (VEGFR)-1, whereas PlGF-2 also binds to a nontyrosine kinase receptor, neuropilin-1, and neither PlGF nor PlGF-3 bind heparin. PlGF preferentially forms heterodimers with VEGF that are mitogenic toward endothelial cells capable of binding with high affinity to both tyrosine kinase receptors VEGF receptors VEGFR-1 and -2 (8). Elevated PlGF expression appears to correlate with pathological angiogenesis, as it has been demonstrated in the vitreous humor of patients with severe diabetic retinopathy (9) and during abnormal placental development (10). Elevated levels of PlGF correlate with increased VEGF expression in the eye (11). This apparent coregulation of PlGF and VEGF in diabetic retinopathy raises the possibility that PlGF could modulate angiogenesis by forming heterodimers with VEGF (8). Loss of PlGF impairs angiogenesis in ischemic retina, limb, and heart, in wounded skin, and in cancer without affecting physiological angiogenesis (12). Moreover, angiogenic therapy with 2959

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suggesting that an important role of PlGF is during vascular morphogenesis (15,16). In contrast, during pathological angiogenesis PlGF appears to play a direct role in promoting vascular growth (12). Many of the angiogenic effects of VEGF have been linked to signaling through phosphatidylinositol 3-kinase (PI3K) and increased expression of Bcl-2 (17) and several other antiapoptotic genes, such as survivin and COX-2 (18). Therefore, we sought to investigate the role of PlGF homodimer and PlGF/VEGF heterodimer in angiogenesis/survival and to determine whether they acted via the PI3K pathway. We demonstrate here that PlGF and PlGF/VEGF induce the formation and long-term maintenance of capillary networks in culture and that this effect occurs in concert with a significant increase in the expression of Bcl-2; both are directly regulated by PI3K signaling. RESEARCH DESIGN AND METHODS

FIG. 1. VEGF, PlGF, and PlGF/VEGF promote endothelial cell survival. A: Endothelial cells were incubated with or without LY294002 (40 ␮mol/l), a potent PI3K inhibitor, and in the presence or absence of VEGF (100 ng/ml) or PlGF (100 ng/ml) or PlGF/VEGF (100 ng/ml) for 24 h. Cell viability was measured using Trypan blue exclusion. LY294002 caused a marked decrease in cell viability compared with growth factor–treated cells. The number of dye-free cells were counted under a phase microscope in five random fields (ⴛ200 magnification) per well. B: Endothelial cells were seeded on type I collagen and exposed for 14 days to VEGF (100 ng/ml), PlGF (100 ng/ml), or PlGF/VEGF (100 ng/ml). Viable cells were counted after Trypan blue exclusion at 2-day intervals up to day 14. PlGF and PlGF/VEGF led to a progressive increase in cell viability that plateaued after day 8. VEGF-induced cell viability declined after day 8. Data are expressed as means ⴞ SD of three independent experiments. *P < 0.05 for PlGF and PlGF/VEGF vs. VEGF.

PlGF was recently found to enhance myocardial angiogenesis with efficiency comparable to that of VEGF, and inhibition of VEGFR-1 blocked angiogenesis in a variety of pathological conditions (13). PlGF-mediated angiogenesis was proposed to result from displacement of VEGF from a “VEGFR-1 sink,” thereby increasing the fraction of VEGF available to activate VEGFR-2 (14). We recently showed that VEGF-mediated VEGFR-1 signaling inhibits VEGFR2–mediated endothelial cell proliferation via nitric oxide (NO) (15), raising the possibility that PlGF could promote angiogenesis by transducing intracellular signals through VEGFR-1. Despite these findings, the mechanisms by which PlGF promotes angiogenesis or survival of endothelial cells remain to be defined. During physiological angiogenesis, VEGFR-1 signaling inhibits VEGFR-2–mediated endothelial cell proliferation, 2960

Reagents. Recombinant human VEGF165 and PlGF-1 were obtained from ReliaTech (Braunschweig, Germany). PlGF/VEGF heterodimer was purchased from R&D System Europe (Abingdon, U.K.). Anti–Bcl-2 polyclonal antibody was from Santa Cruz Biotechnology (Holly Ditch Farm, Calne, U.K.). Anti-Akt and anti–phospho-Akt polyclonal antibodies were obtained from New England Biolabs (Hitchin, Hertfordshire, U.K.). All other materials were from SigmaAldrich (Poole, Dorset, U.K.) unless stated otherwise. Cell culture. Primary cultures of microvascular bovine retinal endothelial cells (BRECs) were isolated from freshly isolated calf eyes by homogenization and a series of filtration steps, as described previously (19). Primary BRECs were cultured in microvascular endothelial cell basal medium (MECBM) with growth supplement (TCS CellWork, Buckingham, U.K.) in fibronectin-coated dishes. Within a week after initial isolation, BRECs were transferred to new fibronectin-coated dishes using a cloning ring. The cells were cultured in 5% CO2 at 37°C, with the medium changed every 3 days. Endothelial cell homogeneity was confirmed by positive immunostaining for anti–factor VIII antibodies, analyzed by confocal microscopy. Peripheral blood monocytes were isolated from buffy coats using gradient centrifugation over Ficoll (histopaque 1077), as previously described, and subsequently plated onto plastic dishes (20). Adenovirus infection. Recombinant adenoviruses encoding wild-type (AdPTEN-WT) or dominant negative mutant phosphatase and tensin homolog on chromosome 10 (PTEN) (AdPTEN-C/S) were generated and serially amplified in human embryonic kidney 293 cells purified on a Caesium Chloride density gradient by ultracentrifugation, as previously described (21). When endothelial cells were nearly confluent, the medium was changed to MECBM containing 2% fetal bovine serum, and viruses were added to the medium at a dilution of 1:1,000 (multiplicity of infection ⬃100). The cells were incubated for 16 h at 37°C, the medium was changed to serum-free MECBM, and the cells were treated as indicated. In vitro migration assay. Chemotaxis of monocytes to PlGF/VEGF was assessed using a modified Boyden’s Chamber (20). Briefly, monocytes (1.5 ⫻ 106/ml) were seeded in the upper chamber and their migration across a polycarbonate filter in response to 10, 50, and 100 ng/ml PlGF/VEGF was investigated. The upper surface of the filter was scraped and filters were fixed and stained with Diff-Quik (Harleco, Gibbstown, NJ). Ten random fields were counted, and the results were expressed as mean ⫾ SE per field. Cell viability assay. Endothelial cells were plated at a density of 5 ⫻ 104 per well on type I collagen– coated 24-well plates in MCDB131 containing 10% FCS. After 24 h, growth media was replaced with serum-free medium (0.1% FCS) for an additional 24 h. Recombinant human VEGF, PlGF, and PlGF/ VEGF were added in the presence or absence of either tumor necrosis factor-␣ (TNF-␣) (100 ng/ml) or PI3K inhibitor LY294002 for defined periods to the quiescent cells. Cell viability was assessed with trypan blue exclusion, and the number of dye-free cells were counted under a phase microscope in five random fields (⫻200 magnification) per well. Apoptosis assay. Apoptosis was evaluated using fluorescein isothiocyanate– conjugated annexin V/propidium iodide assay kit (R&D System Europe, Abingdon, U.K.) based on annexin-V binding to phosphatidylserine exposed on the outer leaflet of the plasma membrane lipid bilayer of cells entering the apoptotic pathway. Cells were treated with or without pan-caspase inhibitor z-Val-Ala-Dl-Asp-fluoromethylketone (z-VAD-fmk, 50 ␮mol/l) for 1 h before the addition of the growth factors (PlGF and PlGF/VEGF) for 7 days in serum-free media. Briefly, cells were collected by EDTA loosening, pelleted by centrifuDIABETES, VOL. 52, DECEMBER 2003

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FIG. 2. PlGF and PlGF/VEGF inhibit endothelial cell apoptosis. A: Endothelial cells were treated with or without pan-caspase inhibitor z-VAD-fmk (50 mmol/l) in the presence or absence of PlGF (100 ng/ml) or PlGF/VEGF (100 ng/ml) for 7 days. Annexin-V/propidium iodide–negative cells were counted as viable cells (lower left quadrant). B: Cell survival is expressed as a percentage of viable cells in the total cell population. Data are expressed as means ⴞ SD of three independent experiments. gation (1,600 rpm for 5 min), washed in PBS, and resuspended in the annexin V incubation reagent in the dark for 15 min before flow cytometric analysis. The analysis of samples was performed using a FACS 440 flow cytometer (Becton Dickinson, Oxford, U.K.) using an argon ion laser. An excitation wavelength of 488 nm was used with fluorescence emission measured at 530 ⫾ 15 nm through fluorescence channel one. A minimum of 10,000 cells per sample were collected using log amplification for fluorescence channel one and linear amplification for forward light scanner and 90° light scatter before being analyzed using in-house software. In vitro coculture angiogenesis assay. In vitro angiogenesis was assessed as formation of capillary-like structures of endothelial cells cocultured with matrix-producing cells. Experimental procedure followed the manufacturer’s protocol provided with the In Vitro AngioKit (TCS Biologicals, Buckingham, U.K.). To measure the formation of the capillary network, the number of connections among three or more capillary-like structures was counted and expressed as the number of capillary connections per field. Four different fields were analyzed per well, and each field was 0.5 mm2. Tube formation assay. BRECs were sandwiched between two layers of type I collagen gels in 24-well plates at 2.5 ⫻ 104 cells/well, as previously described (19). The gel was covered with MCDB131 media containing 1% FCS with or without the growth factors, and in vitro tube formation was assessed for defined periods. In indicated experiments, the BRECs were either left uninDIABETES, VOL. 52, DECEMBER 2003

fected or were infected with adenoviruses encoding catalytic inactive mutant of PTEN (AdPTEN-C/S) or wild-type PTEN (AdPTEN-WT), or empty virus (AdEV) overnight, as previously described (21). Cell cultures were observed under a phase contrast microscope and photographed in five different random fields (⫻100 magnification). The total tube length (mm/mm2) was quantified with a National Institutes of Health image analysis system. Western blot analysis. BRECs were stimulated with growth factors for the indicated times and then processed for immunoblotting as described (19) with antibodies against the following proteins: Bcl-2, Akt, and phospho-Akt. Statistical analysis. All experiments were repeated at least three times, and the results were expressed as means ⫾ SD. Statistical analysis was performed using the one-tailed Student’s t test on log-normalized data, and P ⬍ 0.05 was considered statistically significant.

RESULTS

PlGF and PlGF/VEGF promote endothelial cell viability. Trypan blue exclusion was used to determine whether PlGF or PlGF/VEGF promoted endothelial cell survival via a PI3K-dependent pathway. In serum-free conditions, endothelial cells were exposed to these growth factors in the 2961

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FIG. 3. PlGF and PlGF/VEGF mediate in vitro angiogenesis via VEGFR-1. A: Migration of monocytes to PlGF/VEGF (10, 50, and 100 ng/ml) was performed using a modified Boyden chamber, as described in RESEARCH DESIGN AND METHODS. PlGF/ VEGF heterodimer induced a dose-dependent increase in migration. B: Formation of capillary-like structures of BRECs cultured with matrix-producing cell types was observed after 11 days. Cells were stimulated with PlGF (50 ng/ml) or VEGF (20 ng/ml) in the absence or presence of the anti–VEGFR-1 antibody (100 ng/ml) or sVEGFR-1 (50 ng/ml) and stained using an anti-CD31 antibody. Both PlGF and VEGF induced a twofold increase of capillary network structures, which was inhibited in the presence of anti–VEGFR-1 antibody. Data are means ⴞ SD of three independent experiments. **P < 0.01, ***P < 0.01 vs. control.

presence or absence of the PI3K inhibitor LY294002 at a concentration shown previously to selectively block PI3K activation (22). Both PlGF and PlGF/VEGF promoted cell survival that was comparable with VEGF, and their ability to rescue endothelial cells was inhibited by LY294002 (Fig. 1A). Exposure of endothelial cells to PlGF or PlGF/VEGF for 14 days in serum-free medium resulted in a progressive increase in endothelial cell viability that plateaued after day 8 (Fig. 2B). In contrast, VEGF-induced cell viability declined after day 8, and this difference was statistically significant at days 10 and 12 with respect to PlGF/VEGF (P ⬍ 0.05) and PlGF (P ⬍ 0.05), respectively (Fig. 1B). PlGF and PlGF/VEGF protect endothelial cells against apoptosis. One of the earliest events in the apoptotic cascade is externalization of phosphatidylserine from the inner face of the plasma membrane to the outer cell surface (23). Fluorophore-labeled annexin V (a protein that exhibits nanomolar affinity for phosphatidylserine) binding to externalized phosphatidylserine has been extensively employed as a reliable marker of apoptosis (23). Flow cytometry was performed on annexin-V/propidium iodide–stained cells following treatment with PlGF or PlGF/VEGF for 7 days in the presence or absence of the 2962

pan-caspase inhibitor z-VAD-fmk to determine the survival mechanism. PlGF and PlGF/VEGF inhibited apoptosis compared with control in untreated cells. In addition, both ligands enhanced the survival effect of z-VAD-fmk, suggesting that they inhibit some caspase-independent aspect of apoptosis (Fig. 2A and B). PlGF/VEGF activates VEGFR-1. It is established that human peripheral blood monocytes express only VEGFR-1 (20). PlGF/VEGF stimulated monocyte migration in a concentration-dependent fashion in a modified Boyden’s chamber (Fig. 3A). Similar results were obtained with PlGF and VEGF (data not shown). The chemotactic activity of PlGF/VEGF was abolished by heating the peptide at 95°C for 10 min before addition to the lower well of the Boyden chamber (data not shown). To investigate that activation of VEGFR-1 affects angiogenesis, we used functionally neutralizing anti–VEGFR-1 antibody in an in vitro angiogenesis assay to block VEGF- and PlGF-induced angiogenesis. Quantitative analysis of the number of capillary connections per field showed that the basal formation of capillary-like structures was increased after stimulation with VEGF (20 ng/ml) and PlGF (50 ng/ml) (Fig. 3B). Inhibition of VEGFR-1 using anti–VEGFR-1 DIABETES, VOL. 52, DECEMBER 2003

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FIG. 4. PlGF and PlGF/VEGF promote and prolong in vitro angiogenesis. A: BRECs were sandwiched between two layers of type I collagen in serum-deprived medium in the presence of VEGF (100 ng/ml), PlGF (100 ng/ml), or PlGF/VEGF (100 ng/ml) for up to 14 days. Images of representative microscope fields (ⴛ10): I–II, Control; IV–VI, VEGF; VII–IX, PlGF; and X–XII, PlGF/VEGF. B: Total tube length was measured in each treatment at indicated time points as mm/mm2. VEGF induced a rapid increase in capillary networks that reached a maximum at day 5; however, PlGF had no effect on tube formation until day 5, while PlGF/VEGF sustained in vitro angiogenesis over the entire 14 days. Data are means ⴞ SD or representative of three independent experiments. **P < 0.01, ***P < 0.01 vs. control.

antibody inhibited VEGF- and PlGF-induced capillary connections (P ⬍ 0.001) when compared with VEGF or PlGF alone. The basal capillary network was inhibited by soluble VEGFR-1, which blocks PlGF and VEGF activity, indicating that these growth factors are involved in the process under basal conditions. PlGF and PlGF/VEGF promote sustained in vitro tube formation. BRECs were grown in collagen gel in the presence of VEGF, PlGF, or PlGF/VEGF for up to 14 days in culture. All three growth factors induced angiogenesis, but only PlGF-containing ligands sustained tube formation over this period (Fig. 4A). VEGF, PlGF, and PlGF/VEGF induced in vitro angiogenesis as determined by total tube lengths in a time-dependent manner (Fig. 4B). VEGF induced a rapid increase in capillary networks that reached maximum tube length after 5 days in culture but then dissociated at a rate parallel to the untreated cells over the next 10 days. Unlike VEGF, PlGF had no significant effect on tube formation during the first 5 days of culture, however by day 10 it promoted in vitro angiogenesis. PlGF/VEGF was the most effective promoter of DIABETES, VOL. 52, DECEMBER 2003

capillary-like structures, and it sustained tube formation over the entire 14 days of culture. PlGF and PlGF/VEGF upregulate and maintain Bcl-2 expression. VEGF stimulates expression of Bcl-2 in HUVEC (24). Western blotting analysis revealed that Bcl-2 expression was upregulated following 3 days exposure of endothelial cells to VEGF, PlGF, or PlGF/VEGF; the highest level of expression of Bcl-2 was induced by PlGF/VEGF (Fig. 5). After 14 days, there was a marked decrease in Bcl-2 expression in control and VEGF-treated cells; however, both PlGF- and PlGF/VEGF-treated cells maintained a relatively higher level of Bcl-2 expression. Hence, PlGF and PlGF/VEGF, but not VEGF, were able to sustain increased Bcl-2 expression over 14 days. Sustained tube formation by PlGF requires activation of the PI3K pathway. To investigate the role of the PI3K pathway in PlGF ligand–induced tube formation, in vitro angiogenesis was assessed following overexpression of either wild-type (AdPTEN-WT) or dominant negative mutant PTEN (AdPTEN-C/S). AdPTEN-WT hydrolyzes the phospholipid products of PI3K and has been shown to 2963

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DISCUSSION

FIG. 5. PlGF and PlGF/VEGF upregulate and maintain Bcl-2 expression. Western blots of whole-cell lysates from BRECs cultured on type I collagen gel treated with VEGF (100 ng/ml), PlGF (100 ng/ml), or PlGF/VEGF (100 ng/ml) at 100 ng/ml over 3 and 14 days were blotted with anti–Bcl-2 antibody. VEGF, PlGF, and PlGF/VEGF induced expression of Bcl-2 at day 3; however, only PlGF and PlGF/VEGF maintained relatively higher levels of Bcl-2 expression at day 14. Data are from representative experiments.

inhibit VEGF-mediated signaling and function (21). Endothelial cells were infected with recombinant adenoviruses encoding wild-type (WT) or catalytically inactive PTEN (C/S) or with a control, empty adenovirus (AdEV). After 7 days in culture, uninfected and AdEV-infected cells formed an organized network of endothelial tubes (data not shown). In contrast, AdPTEN-WT overexpression markedly inhibited VEGF-mediated tube formation (data not shown), consistent with previous results (21). Tube formation induced by PlGF and the PlGF/VEGF was also attenuated by AdPTEN-WT (Fig. 6A and B), indicating that PI3K is required for the angiogenic effects of these two ligands. As in previous experiments, PlGF and PlGF/VEGF both sustained tube formation for up to 14 days, whereas VEGF-mediated tubes had dissociated by this time (Fig. 6B and C). This defect in VEGF-mediated tube formation was rescued by the dominant negative PTEN mutant, suggesting that sustained angiogenesis requires prolonged PI3K activity (Fig. 6C). Moreover, after 14 days of culture, tube length induced by PlGF or PlGF/VEGF in uninfected and AdEV-infected cells was similar to that seen in the presence of AdPTEN-C/S (Fig. 6C). We hypothesized that overexpression of wild-type PTEN will suppress Bcl-2 expression, whereas inactive PTEN will upregulate it in response to these growth factors. BRECs were infected with or without AdPTEN-WT, AdPTEN-C/S, or AdEV and observed over 7 days. Compared with AdEV or AdPTEN-WT–infected cells, overexpression of AdPTEN-C/S dramatically upregulated Bcl-2 expression after treatment with the growth factors (Fig. 6D). Taken together, these data suggest that PTEN is a potent inhibitor of PlGF- and PlGF/ VEGF-mediated angiogenesis and that inactivation of PTEN upregulates Bcl-2 expression. Akt is an important downstream target of PI3K, as it regulates PI3K-mediated cell survival. To determine whether the effects of PlGF on the survival of BRECs correlated with effects on Akt activation, Akt phosphorylation was examined in whole-cell lysates treated with or without growth factors (VEGF, PlGF, and PlGF/VEGF). PlGF induced a rapid increase in Akt phosphorylation that peaked at 15 min, while VEGF and PlGF/VEGF induced maximal activation of Akt between 30 and 60 min (Fig. 6E). These results suggest that activation of PI3K is required for PlGF-mediated endothelial cell survival. 2964

Although VEGF is the major factor in the initiation of advanced stages of diabetic retinopathy, it is increasingly recognized that PlGF is a significant factor in promoting the aberrant angiogenesis characteristic of a variety of pathological states. The mechanisms by which PlGF modulates pathological angiogenesis are still poorly understood. This study demonstrates that both PlGF homodimers and PlGF/VEGF heterodimer promote and sustain in vitro tube formation and BRECs survival in a PI3K dependent manner. Notably, this sustained endothelial cell survival was not observed following treatment with VEGF homodimers. Like VEGF, both PlGF and PlGF/VEGF upregulated the antiapoptotic protein Bcl-2, but unlike VEGF, increased Bcl-2 expression was maintained for up to 14 days, coincident with these factors’ ability to sustain in vitro tube formation. Our studies are consistent with the ability of adenoviral PlGF gene transfer studies in the ear of nude mice that resulted in the formation of large vessels that persisted for ⬎14 days (13). Reports by Cao et al. (8) and Birkenhager et al. (25) demonstrated that VEGF and PlGF can form heterodimer in Escherichia coli; both genes were expressed as unfused genes, and the resultant heterodimers showed mitogenic activity. Remarkably, we found that PlGF/VEGF was more effective at retaining in vitro tube formation of BRECs in prolonged culture than PlGF. Moreover, monocyte migration induced by PlGF/VEGF indicates that PlGF/VEGF activates VEGFR-1. Thus the ability of neutralizing anti–VEGFR-1 antibody to dramatically reduce the number of capillary connections in the in vitro angiogenesis assay indicates that VEGFR-1 ligands promote branching angiogenesis, and this is consistent with the increased vascular branching induced by PlGF overexpression (26). Importantly, this suggested that PlGF could enhance its angiogenic effects through dimerization with VEGF. It will be of significant interest to determine whether PlGF/VEGF occurs in vivo like VEGF and PlGF in proliferative diabetic retinopathy (9,11). Formation of functional receptor heterodimers have been observed in many multireceptor systems, such as those recognizing fibroblast growth factors (27), PDGF (28), and VEGF (29). It is known that VEGF, PlGF, and VEGF/PlGF heterodimer can bind to VEGFR-1, whereas VEGF can also bind to VEGFR-2. The VEGFR-1/VEGFR-2 heterodimer may have different signaling properties to the homodimers of each type of receptor. Our data indicate that the VEGFR-1– containing heterodimer may be important in prolonging angiogenesis as PlGF and PlGF/VEGF induced tube formation as well as Bcl-2 expression that was sustained over 14 days. In contrast, VEGFR-2– containing homodimers appear to be involved in initiating angiogenesis but not sustaining it. Such differential signaling has been demonstrated for the PDGF ␣ and ␤ receptors, which form homo- and heterodimers dependent on whether both receptor types are expressed in the same cells and dependent on the PDGF isoform (30). Bcl-2 has been reported to play a crucial role in the inhibition of apoptosis via the inhibition of caspases (31,32). VEGF induces expression of Bcl-2 (17). In the present study, there was a marked decrease in Bcl-2 expression in the control and VEGF-treated cells, however, both PlGF- and PlGF/VEGF-treated BRECs mainDIABETES, VOL. 52, DECEMBER 2003

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FIG. 6. PlGF- and PlGF/VEGF-mediated sustained angiogenesis require activation of PI3K/PTEN signaling pathway. Subconfluent BRECs were uninfected (Un) or infected with recombinant adenoviruses encoding catalytic inactive PTEN (AdPTEN-C/S), AdPTENWT, or an empty virus (AdEV) for 16 h at 37°C. The infected cells were then sandwiched between two layers of type I collagen gels and treated with VEGF (100 ng/ml), PlGF (100 ng/ml), or PlGF/ VEGF (100 ng/ml) over a period of 14 days. A: Images of representative microscope fields (ⴛ10). B and C: Total tube length was measured in each treatment at days 7 and 14 as mm/mm2. D: Adenovirus-infected BRECs cultured on type I collagen gel treated with VEGF (100 ng/ml), PlGF (100 ng/ml), or PlGF/VEGF (100 ng/ml) over 7 days. The cells were then lysed and resolved by SDS-PAGE and immunoblotted with anti–Bcl-2 antibody and normalized against ␣-tubulin. E: BRECs were treated with VEGF (25 ng/ml), PlGF (25 ng/ml), or PlGF/VEGF (50 ng/ml) as indicated. After lysis, equal amounts of protein were resolved by SDS-PAGE and then immunoblotted with anti–phospho-Akt (Ser473) and anti-Akt. Data are means ⴞ SD or representative of three independent experiments. Day 3 (B); Day 7 (C); Day 14 (D). *P < 0.05, **P < 0.01 vs. AdEV; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. AdPTEN-C/S.

tained a relatively higher level of Bcl-2 expression over the 14-day period. Furthermore, both PlGF and PlGF/VEGF significantly inhibited apoptosis, as indicated by a reduction in annexin V–positive cells. Interestingly, these growth factors enhanced the survival effect of z-VAD-fmk, suggesting that they can inhibit some caspase-independent aspect of apoptosis. Recent studies have identified caspase-independent apoptosis pathways, Bax- or Mtdinduced cell death, that are not blocked by caspase DIABETES, VOL. 52, DECEMBER 2003

inhibitors (33,34). It is possible that VEGFR-1 ligands may inhibit these pathways, and further studies on their relationship are necessary. However, in the presence of the PI3K inhibitor, neither growth factor was able to promote survival of endothelial cells. As the phospholipid second messengers generated by PI3K provide a common mechanism for multiple steps during angiogenesis (21), the intracellular mechanism of PlGF-induced vessel growth was studied following modu2965

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FIG. 7. Diagram of the regulatory pathways mediated by VEGF receptors homo- and heterodimers in BRECs during physiological or pathological angiogenesis.

lation of PI3K signaling by overexpression of PTEN constructs in cultured BRECs. AdPTEN-C/S, which acts as a dominant negative mutant to block the dephosphorylation of PI 3,4,5-triphosphate enhanced in vitro tube formation and upregulated PlGF- and PlGF/VEGF-mediated Bcl-2 expression. The loss of PTEN results in increased 3-phosphoinositides and downstream activation of Akt (35–37). Active Akt modulates a number of downstream targets that affect apoptosis, cell cycle progression, and angiogenesis (38,39). Activated Akt can activate the cAMP response element binding protein (40,41). cAMP response element binding protein can induce Bcl-2 expression in a myriad of cell types (39,42,43). Indeed, both PlGF and PlGF/VEGF stimulated Akt phosphorylation in cultured BRECs, indicating that the prosurvival activity of these growth factors requires PI3K/Akt signaling, as is the case with VEGF. Taken together, these findings indicate that VEGFR-1 is involved in endothelial cell differentiation, and its activation by its selective ligands leads to the formation of blood vessels that persisted over long duration. This notion is supported by a recent study in which transgenic overexpression of PlGF in the skin using the keratin-14 promoter led to substantial increases in the number, branching, and size of dermal blood vessels (44). Based on the present study, we cannot entirely rule out the possibility that the delayed effects of PlGF were mediated in part by the upregulation of some other factor(s), possibly even VEGF. However, increasing evidence suggests that PlGF can directly regulate key aspects of vascular growth. A recent study demonstrated that PlGF stimulates VEGFR-1–mediated angiogenesis and collateral growth in ischemic heart and limb tissue with an efficiency that is at least comparable to that of VEGF (13). PlGF was also shown to induce smooth muscle cell migration in vitro, and VEGFR-1 is expressed on smooth muscle cell migrations and peri2966

cytes (45). Consistent with this idea, K14-PlGF mice displayed an increase in vascular density with predominantly smooth muscle– coated vessels, while PlGF knockout mice had a high proportion of vessels that lacked perivascular cells (44). It is important to note that PlGF induces NO production from endothelial cells and that NO is a cue for endothelial cell differentiation (15). The in vitro findings of this study support our notion that PlGF mediates neovessel maturation and stabilization not only by recruiting smooth muscle cell migrations and pericytes to the vascular endothelium, but also by acting directly on endothelial cells per se and upregulating the survival signals. In addition, PlGF may enhance its angiogenic effect on endothelial cell through synergism or dimerization with VEGF. VEGF is a key survival factor (24,47,48) that is increased in diabetic retinopathy (11,46). However, increased PlGF levels were only observed in proliferative diabetic retinopathy, which is a feature of retinal neovascularization (9). As VEGF could not sustain tube formation and survival effect for as long as PlGF or PlGF/VEGF, it is likely that PlGF-containing ligands are the culprit. A model for PlGF and VEGF homo- and heterodimer action during physiological or pathological angiogenesis through the regulatory pathways mediated by VEGFR-1 and -2 is proposed (Fig. 7). Our findings suggest that the ability of PlGF and PlGF/VEGF to enhance endothelial cell survival may come from their capacity to sustain angiogenesis and upregulate the antiapoptotic protein Bcl-2 via the PI3K pathway, thus contributing to the aberrant vascular growth observed during pathological angiogenesis. ACKNOWLEDGMENTS

This study was supported by grants from the Wellcome Trust 053347/B/98/Z (M.B. and A.A.) and British Heart Foundation Program Grant RG98003 (A.A.). DIABETES, VOL. 52, DECEMBER 2003

J. CAI AND ASSOCIATES

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