bFGF and VEGF synergistically enhance endothelial cytoprotection via decay-accelerating factor induction

Justin C. Mason, Elaine A. Lidington, Saifur R. Ahmad, and Dorian O. Haskard

British Heart Foundation Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College School of Science, Technology and Medicine, Hammersmith Hospital, London W12 ONN, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
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The complement-regulatory protein decay-accelerating factor (DAF) can be upregulated on endothelial cells (EC) by protein kinase C (PKC)-dependent and -independent pathways. We hypothesized that basic fibroblast growth factor (bFGF) might induce EC DAF expression, providing a cytoprotective mechanism for angiogenic neovessels against complement-mediated injury. Incubation of umbilical vein, aortic, and dermal EC with bFGF or vascular endothelial growth factor (VEGF) significantly increased DAF expression. Growth factor-induced EC proliferation was inhibited by PKC antagonists. In contrast, although PKC antagonists inhibited VEGF-induced DAF expression, bFGF-induced DAF was unaffected. Investigation of mitogen-activated kinase (MAPK) pathways also revealed differences, with bFGF-induced DAF dependent on p44/42 and p38 MAPK and VEGF requiring activation of p38 MAPK alone. Upregulation of DAF by bFGF was functionally relevant, reducing C3 deposition on EC after complement activation by 60% and resulting in marked reduction in complement-mediated EC lysis. bFGF and VEGF were synergistic in terms of DAF expression, resulting in enhanced cytoprotection. These observations reveal parallel PKC-dependent and -independent pathways regulating complement activation during angiogenesis. Further elucidation of these pathways may provide important insights into innate cytoprotective mechanisms in endothelium.

complement; complement-mediated injury; angiogenesis


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INTRODUCTION
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THE COMPLEMENT SYSTEM IS a highly complex cascade of enzymes and regulatory proteins (49). In addition to its critical role in host defense against microorganisms, complement exerts an important modulatory influence over inflammatory responses. However, as a consequence of its membrane actions, activation of the complement cascade may also result in bystander activation or cytolysis of host tissues (30). Thus complement activation has been implicated in a variety of chronic inflammatory disease states, including atherosclerosis and rheumatoid arthritis (32, 37, 46). To counteract the deleterious effects of complement, nucleated cells have developed an array of membrane-bound and soluble regulators of complement activation (21, 31). These factors utilize distinct mechanisms for the control of complement and may act cooperatively. Decay-accelerating factor (DAF, CD55), which prevents the formation and accelerates the decay of C3 and C5 convertases (22), membrane cofactor protein (MCP, CD46), which binds C3b and C4b and facilitates their degradation by factor I, and CD35, which performs both of these functions, operate at the proximal end of the cascade (21). In contrast, CD59 serves to inhibit the membrane attack complex (MAC, C5b-C9) by inhibiting the incorporation of C9 (31). In addition, fluid-phase regulators of complement activation including factor H, factor I, and clusterin are important in cytoprotection (31).

The membrane-bound regulators of complement activation DAF, MCP, and CD59 are expressed by endothelial cells (EC) and contribute to the control of complement activation on the cell surface. However, the precise contributions of each of the molecules to the protection of EC, both in the resting state and during inflammation, remain to be determined (6). In our previous work (20, 24, 26) we showed that DAF, but not MCP or CD59, is inducible on the EC surface after stimulation with a number of agonists acting through distinct signaling pathways. Thus DAF is inducible on EC in response to tumor necrosis factor (TNF)-alpha , interferon (IFN)-gamma , and the C5b-9 MAC via a protein kinase C (PKC)-independent pathway, whereas thrombin- and vascular endothelial growth factor (VEGF)-induced DAF upregulation is PKC dependent. Furthermore, the upregulation of DAF expression by these mediators protects EC from complement-mediated lysis, suggesting that DAF upregulation may be important for maintaining vascular integrity during subacute and chronic inflammatory responses involving complement activation (20, 24, 26). Moreover, the ability of DAF to protect host endothelium against complement-mediated injury has been confirmed in vivo with xenotransplantation models (28).

It is now clear that angiogenesis plays a central role in the tissue remodeling associated with chronic inflammatory diseases (11). Recent studies have harnessed this observation by utilizing proangiogenic factors such as VEGF and basic fibroblast growth factor (bFGF, FGF-2) for the promotion of new vessel growth during therapeutic angiogenesis (17). Because vascular endothelium in chronic inflammation may be continuously exposed to sublytic levels of activated complement components, new blood vessels are at risk of complement-mediated injury and C5b-C9-induced cellular activation (18, 30, 32, 38, 46). This led us to the hypothesis that the proangiogenic growth factors bFGF and VEGF, in addition to inducing angiogenesis, may condition endothelium against vascular injury through the induction of one or more of the complement-regulatory proteins DAF, MCP, and CD59. In the setting of therapeutic angiogenesis, such an effect would help maintain neovessel integrity in the presence of low-grade complement activation (32, 46).

Although bFGF and VEGF are both heparin-binding growth factors capable of inducing angiogenesis in vitro and in vivo, important differences exist between the two. VEGF is secreted, and its actions are largely confined to EC and only a few other cells of nonendothelial origin (10). In contrast, bFGF may be secreted or membrane bound and is closely associated with extracellular matrix. The actions of bFGF are more diverse than those of VEGF, involving a wider range of nonendothelial cells and including important effects on organ development and differentiation, in addition to angiogenesis (5). Synergistic effects of the two growth factors have been demonstrated with in vitro assays of angiogenesis in three-dimensional gels (15, 36) and in vivo in a unilateral hindlimb ischemia model of angiogenesis (1).

In this study, we show that bFGF and VEGF stimulate DAF expression on the surface of human EC and, when combined, exert a synergistic effect. The increased expression of DAF leads to reduced complement C3 binding and results in enhanced protection against EC lysis. These data suggest that in addition to their proangiogenic effects, bFGF and VEGF may also induce important cytoprotective mechanisms against complement-mediated vascular injury.


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Monoclonal antibodies and other reagents. The monoclonal antibodies (MAbs) A35 (IgG1) against CD59 and TRA-2-10 (IgG1) anti-MCP were gifts from Prof. B. P. Morgan (University of Wales, Cardiff, UK) and Prof. J. P. Atkinson (Washington University School of Medicine, St Louis, MO), respectively. The anti-DAF MAbs used included MAbs 1H4 (IgG1) and 5B2 (IgG1), which were gifts from Dr. D. Lublin (Washington University School of Medicine) and Dr. T. Fujita (University of Tsukuba, Tsukuba, Japan), respectively. The anti-endoglin (CD105) MAb RMAC8 (IgG2a) was a gift from Dr. A. d'Apice (St. Vincent's Hospital, Victoria, Australia). Recombinant human VEGF 165 and the neutralizing anti-VEGF polyclonal antibody were purchased from Pepro Tech EC (London, UK). Recombinant human bFGF and MAb against bFGF were from R&D Systems (Abingdon, UK). Bovine serum albumin (BSA), nonenzymatic cell dissociation solution, and cycloheximide (CHX) were purchased from Sigma (Poole, UK). The PKC antagonists Ro-31-8220 and bisindolylmaleimide 1, the cell-permeable selective mitogen-activated protein kinase (MAPK) kinase (MEK)-1 inhibitor PD-98059, which inhibits activation of p44/42 MAPK, and the p38 MAPK inhibitor SB-202190 were purchased from Calbiochem (Nottingham, UK). Normal human serum (NHS) was obtained from samples of blood taken from healthy volunteers and prepared as described previously (26).

Cell isolation and culture. Human umbilical vein EC (HUVEC) and dermal microvascular EC (DMEC) were isolated and cultured as described previously (27, 51). Human aortic EC (HAEC) were purchased as growing cultures from Promocell (Heidelberg, Germany) and were maintained in EC growth medium supplemented with endothelial growth supplement (0.4%), 5% fetal bovine serum (FBS), epidermal growth factor (10 ng/ml), hydrocortisone (1 µg/ml), amphotericin B (50 ng/ml), and gentamicin (50 µg/ml) (all from Promocell). For all experiments, the EC culture medium was changed to M199 (ICN Biomedicals, Costa Mesa, CA) supplemented with 10% FBS (Hyclone Laboratories, Logan, UT), 100 IU/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine (all from GIBCO BRL Life Technologies, Paisley, UK).

Flow cytometry. Monolayers of EC were harvested by exposure to trypsin-EDTA (ICN) and analyzed by flow cytometry with an Epics XL-MCL flow cytometer (Coulter, Hialeah, FL) as described previously in detail (26). In experiments involving pharmacological antagonists, each inhibitor was added 60 min before the addition of growth factors. In some experiments the results are expressed as the relative fluorescence intensity (RFI), which represents the mean fluorescence intensity (MFI) with test MAb divided by the MFI using an isotype-matched irrelevant MAb. In all experiments cell viability was assessed by examination of EC monolayers before staining with phase-contrast microscopy, cell counting, and estimation of trypan blue exclusion.

Northern blotting analysis. bFGF-stimulated or unstimulated EC were lysed in guanidium isothiocyanate (Sigma), and RNA was extracted as described by Chomczynski and Sacchi (8). Purified RNA was resuspended in 20 µl of RNase-free water and stored at -70°C before use. The probe for DAF (29) was provided by Prof. B. P. Morgan (University of Wales). The technique used for Northern blotting was previously described in detail (26). Northern blots were quantified by an Appligene image analysis system (Appligene, Durham, UK), and densitometry was performed with NIH Image program 1.52 software. Values were corrected with respect to ethidium bromide-stained rRNA loading patterns, and an arbitrary value of 1 was assigned to unstimulated EC.

Cell proliferation and bromodeoxyuridine double staining assays. HUVEC (5 × 103 cells per well) were cultured in M199-10% FBS (plain medium) for 24 h in 96-well plates in the absence of growth factors. The medium was then replaced with plain medium alone or medium containing bFGF (20 ng/ml) or VEGF (25 ng/ml). The cells were then incubated for a further 72 h, with 3H-labeled thymidine (1 µCi/ml; Amersham Pharmacia Biotech, Little Chalfont, UK) added 16 h before the end of the assay. EC were then harvested, and proliferation was quantified with an automated Betaplate 96-well harvester (Wallac Oy, Turku, Finland) as previously described (24). In the bromodeoxyuridine (BrdU) incorporation experiments subconfluent EC were treated with 20 ng/ml bFGF for 48 h, with BrdU (final concentration 20 µM) added 16 h before the end of the assay. After harvesting the cells were resuspended in ice-cold PBS and fixed in 70% ethanol at 4°C. After centrifugation at 500 g and washing in PBS-1% BSA, 0.2 M HCl was slowly added to the EC while a vortex was maintained. After 10 min and centrifugation at 500 g, 1 ml of 0.1 M Na2B4O7 · 10H2O, pH 8.5, was added to neutralize the acid. EC were washed in PBS-1% BSA-0.5% Tween 20 and transferred to 96-well V-bottom plates, and primary antibodies were added for 30 min at 4°C. After two washes in PBS-1% BSA, biotinylated F(ab')2 rabbit anti-mouse Ig was added for 30 min at 4°C, followed by washing as above and incubation with streptavidin-phycoerythrin (Vector Laboratories, Burlingame, CA) and FITC-conjugated anti-BrdU MAb (Becton Dickinson, Mountain View, CA) with 10% mouse serum for 30 min at 4°C. EC were analyzed on a Becton Dickinson FACScan flow cytometer.

C3 binding and cell lysis assays. The methods used for detection of cell surface C3 and calcein-mediated lysis were described in detail previously (24). Briefly, EC were cultured in the presence and absence of bFGF (20 ng/ml) or VEGF (25 ng/ml) for 48 h and then opsonized with anti-endoglin MAb RMAC8 and incubated with 100 µl of 20% NHS in M199 for 3 h at 37°C before analysis of C3 deposition by flow cytometric analysis (24). In the inhibition studies, blocking MAbs were added to the assay with RMAC8 to achieve a concentration of 25 µg/ml. In the cell lysis experiments EC pretreated with bFGF, VEGF, or plain medium alone for 48 h were loaded with calcein-acetoxymethyl ester (AM) (Molecular Probes, Leiden, The Netherlands), opsonized with MAb RMAC8, and then incubated with 5-10% baby rabbit complement (Serotec, Oxford, UK) for 30 min at 37°C. Calcein release into the supernatant was estimated using a CytoFluor 2300 fluorescence plate reader (Millipore, Bedford, MA) as previously described (24). Percent specific lysis in triplicate wells was calculated as (complement-mediated release - spontaneous release)/(maximal release - spontaneous release) × 100%, where maximal release = complement-mediated release + detergent-mediated release.

Anti-bFGF and anti-VEGF antibody crossover experiments. In the antibody crossover experiments, monolayers of EC were stimulated for 48 h with bFGF (20 ng/ml) or VEGF (25 ng/ml) in the presence or absence of either anti-bFGF or anti-VEGF neutralizing antibodies (50 µg/ml). After harvesting, single-cell suspensions were analyzed by flow cytometry for the expression of DAF with FITC-labeled anti-DAF MAb 67 (Serotec).

Statistics. Differences between the results of experimental treatments were evaluated by the Mann-Whitney U-test. To obtain statistical significance of synergy a chi 2-test was used. Differences were considered significant at P values of <0.05.


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bFGF induces DAF expression on human EC. HUVEC and DMEC express significant amounts of DAF on their surface in vitro (2, 26). However, immunohistological analysis of normal human skin showed little DAF expression by vascular EC (25). These observations led us to propose that DAF expression on cultured EC might be a consequence of cell proliferation and that the molecule might be regulated by growth factors. To this end we recently showed (24) that VEGF is capable of inducing DAF expression on cultured EC. To explore this hypothesis further we tested the effects on EC DAF expression of bFGF, another potent proangiogenic factor.

As shown in Fig. 1A, a low level of constitutive DAF expression was present on untreated cells, whereas bFGF led to a significant unimodal increase in the expression of cell surface DAF (Fig. 1). This upregulation of DAF was dose dependent, with maximal expression occurring with 10-20 ng/ml of bFGF (Fig. 1B). In 10 separate experiments bFGF upregulated DAF between two- and fourfold above the basal level on unstimulated HUVEC [RFI (±SD) of unstimulated HUVEC: 21.2 ± 8.7, bFGF-stimulated HUVEC: 65.5 ± 21.8; P < 0.01]. In contrast, bFGF did not increase the expression of either MCP or CD59 above constitutive levels on growth factor-depleted EC (Fig. 1B). Similar experiments were performed on DMEC, because these may more accurately represent those EC of the microvasculature that are involved in angiogenesis, and on HAEC, to represent a vascular bed affected by atherosclerosis. DMEC and HAEC behaved similarly to HUVEC in terms of DAF upregulation in response to bFGF [RFI of unstimulated DMEC: 25.2 ± 2.6, bFGF-stimulated DMEC: 45.2 ± 0.6 (P < 0.05); RFI of unstimulated HAEC: 26.1 ± 1.1, bFGF-stimulated HAEC: 46.6 ± 2.1 (P < 0.05)], with no change in MCP or CD59 observed (data not shown); hence, HUVEC were used for the remaining experiments.


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Fig. 1.   Analysis of decay-accelerating factor (DAF), membrane cofactor protein (MCP), and CD59 expression on human umbilical vein endothelial cells (HUVEC) after stimulation by basic fibroblast growth factor (bFGF). The expression of DAF was assessed by flow cytometry with monoclonal antibody (MAb) 1H4. A: DAF expression on HUVEC is shown in the absence of growth factors [mean fluorescence intensity (MFI) 9.5; open histogram with solid line] and after 48-h stimulation with bFGF (20 ng/ml, MFI 47.7; filled histogram). Background fluorescence (irrelevant antibody and FITC-labeled rabbit anti-mouse Ig) is shown by the open histogram with dotted line. B: EC were stimulated for 48 h with bFGF at varying concentrations before harvesting and analysis by flow cytometry with MAbs 1H4 (anti-DAF), A35 (anti-CD59), and TRA-2-10 (anti-MCP). Bars represent relative fluorescence intensity (RFI) ± SD (n = 3) derived by dividing the MFI with test MAb by the MFI with isotype-matched irrelevant MAb. The figure is representative of 3 similar experiments.

To determine the kinetics of DAF induction by bFGF, HUVEC were cultured in the presence of bFGF for up to 72 h. As shown in Fig. 2A, upregulation was detectable at 24 h and was maximal 48-72 h after stimulation. Analysis of MCP or CD59 on the same cells at these time points demonstrated no change in expression (data not shown). To determine whether an increase in DAF expression was confined to HUVEC that were actively proliferating in response to bFGF, subconfluent cultures were treated with bFGF for 30 h and then pulsed with BrdU for a further 16 h. The cells were then double-stained for DAF and BrdU and examined by dual-color flow cytometry. In three separate experiments there was no difference in DAF expression between BrdU-positive cells [DAF MFI (± SE): 251.4 ± 26.8] and BrdU-negative cells (DAF MFI: 247.5 ± 33.7) (Fig. 2B), suggesting that DAF expression in this context is not dependent on the stage of the cell cycle. The higher RFI in these data compared with those shown in Fig. 1 reflect the use of a different flow cytometer.


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Fig. 2.   Upregulation of DAF expression by bFGF on HUVEC. A: time course for bFGF-induced DAF expression on HUVEC as detected by flow cytometry. The results are presented as RFI ± SD (n = 3). B: HUVEC plated at subconfluence were stimulated for 48 h with bFGF (20 ng/ml), and bromodeoxyuridine (BrdU; 20 µM) was added for the final 16 h of incubation. After harvesting, fixation, and permeabilization of EC, BrdU incorporation and DAF expression were assessed by flow cytometry. Bars show means ± SD (n = 3). The figure is representative of 3 similar experiments.

Comparison of role of PKC in bFGF- and VEGF-induced DAF expression on HUVEC. In recent studies we demonstrated (20, 24, 26) that DAF expression by EC can be mediated by PKC-dependent and -independent pathways. To investigate whether PKC is involved in the bFGF-mediated stimulation of DAF expression, we used the PKC antagonists Ro-31-8220 and bisindolylmaleimide I. The concentration of PKC antagonists used was established in earlier studies in which we observed complete inhibition of phorbol ester-induced effects on EC (26, 27). As shown in Fig. 3A, although pretreatment of EC for 30 min with Ro-31-8220 (0.75 µM) before stimulation with bFGF led to significant inhibition of EC proliferation, this was incomplete. In contrast, the increased proliferation seen with VEGF, although characteristically less than that seen with bFGF, was completely inhibited by Ro-31-8220 (Fig. 3A). Furthermore, although VEGF-induced DAF expression was PKC dependent, that seen in response to treatment with bFGF was not inhibited by Ro-31-8220 (Fig. 3B). Similar results were also obtained when PKC was inhibited with bisindolylmaleimide I (5 µM; not shown). Thus the proliferative effect of bFGF is partially inhibited by PKC antagonists (12), whereas bFGF DAF expression appears to be independent of PKC activation. In contrast, the effects of VEGF on EC DAF expression and proliferation are both PKC dependent (24, 52).


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Fig. 3.   Effect of the protein kinase C (PKC) antagonist Ro-31-82220 on bFGF-induced EC proliferation and DAF upregulation. A: HUVEC were cultured in 96-well microtiter plates (5,000 EC/well) in M199 medium with 10% FBS and no EC growth factor (ECGF) for 24 h before the addition of VEGF (25 ng/ml) or bFGF (20 ng/ml). They were then cultured for a further 72 h before harvesting. 3H-labeled thymidine (1 µCi/ml) was added 16 h before the end of the assay, and the plates were read on an automated plate reader. In test wells, EC were preincubated with Ro-31-8220 (0.75 µM) for 30 min before addition of VEGF/bFGF. Data are expressed as the uptake of [3H]thymidine in counts per minute (mean ± SD, n = 4). B: HUVEC were treated with Ro-31-8220 (0.75 µM) or plain medium alone for 30 min before the addition of VEGF (25 ng/ml) or bFGF (20 ng/ml) and cultured for a further 48 h. After harvesting, EC were analyzed by flow cytometry for the expression of DAF with MAb 1H4, and the data are expressed as percentages of the DAF expression on bFGF/VEGF-treated cells, and bars show means ± SE from 4 experiments on separate EC cultures. *P < 0.02; **P < 0.002.

bFGF and VEGF utilize distinct MAPK signaling pathways for DAF upregulation. To define the signaling pathways involved in the regulation of DAF expression by bFGF further, we used the MEK-1 inhibitor PD-98059, which prevents p44/42 MAPK phosphorylation, and SB-202190, which is an inhibitor of p38 MAPK. After dose titration experiments to determine the optimal concentrations (24), HUVEC were preincubated with PD-98059 (75 µM) or SB-202190 (25 µM) for 1 h before the addition of bFGF. As shown in Fig. 4A, both agents led to a significant inhibition of DAF upregulation on EC (P < 0.05). Moreover, pretreatment with a combination of PD-98059 and SB-202190 led to complete abrogation of bFGF-induced DAF expression (P < 0.01). Both agents, and particularly SB-202190, induced a change in shape of EC but did not lead to any demonstrable cytotoxicity at the concentrations used. Thus EC remained >90% viable, as assessed by cell counting and trypan blue exclusion on the EC populations and also by flow cytometric analysis of CD31 expression (not shown). Because VEGF-induced DAF expression was previously found to be dependent on p38 but not p44/42 MAPK (24), these data demonstrate further the differences between bFGF- and VEGF-induced pathways for DAF upregulation. Furthermore, in contrast to the regulation of DAF, proliferation in response to either bFGF or VEGF was inhibited in a dose-dependent manner by either PD-98059 or SB-202190 (Fig. 4; Ref. 24).


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Fig. 4.   Role of p38 and p42/44 mitogen-activated protein kinase (MAPK) activation in bFGF-induced DAF expression. A: EC were preincubated with SB-202190 (25 µM) or PD-98059 (75 µM) for 60 min before the addition of bFGF (20 ng/ml). Cells were then cultured for a further 48 h. After harvesting, DAF expression was measured by flow cytometry with MAb 1H4. The data are expressed as percentages of the DAF expression on bFGF-treated cells and are presented as means ± SE from 3 experiments on separate EC lines. B and C: HUVEC were cultured in 96-well microtiter plates (5,000 EC/well) in M199 medium with 10% FBS and no ECGF for 24 h before the addition of bFGF (20 ng/ml) and culture for a further 72 h before harvesting. [3H]thymidine (1 µCi/ml) was added 16 h before the end of the assay, and the plates were read on an automated plate reader. In test wells EC were preincubated with PD-98059 (B) or SB-202190 (C), at the concentrations shown, for 60 min before the addition of bFGF (20 ng/ml). Data are expressed as the uptake of [3H]thymidine in counts per minute (mean ± SD, n = 4).*P < 0.05; **P < 0.01.

bFGF-induced DAF follows increase in steady-state mRNA and de novo protein synthesis. Northern analysis was performed with mRNA extracted from unstimulated HUVEC and cells stimulated with bFGF for up to 24 h. As shown in Fig. 5A, two DAF mRNA transcripts (2.4 and 1.8 kb; Ref. 7) were detected at a low level in unstimulated EC. After bFGF stimulation, an increase in DAF mRNA was first detectable at 3 h and maximal at 12 h and declined to basal levels by 24 h after stimulation. Quantification of mRNA levels in resting and bFGF-stimulated EC with densitometric scanning of the 2.4-kb band demonstrated a 3.5-fold increase above baseline at 12 h after stimulation (Fig. 5B). To investigate whether the effect of bFGF on DAF gene transcription was direct or required synthesis of a transactivating factor, EC were preincubated for 30 min with CHX before the addition of bFGF. Incubation with CHX alone led to a superinduction of steady-state DAF mRNA (Fig. 5A). In addition, CHX prevented the increase in steady-state DAF mRNA seen in response to bFGF (Fig. 5A), suggesting that the changes in DAF gene transcription observed are indirect and dependent on the synthesis of one or more intermediary proteins. This was in contrast to the direct effect we observed with thrombin, which induced an increase in DAF mRNA under these conditions (20). To determine whether the increase in DAF on the EC surface observed after bFGF stimulation was dependent on de novo protein synthesis, HUVEC were pretreated with CHX before the addition of bFGF. As shown in Fig. 5C, CHX led to a complete abrogation of bFGF-induced DAF (P < 0.001). Thus the upregulation of DAF expression after stimulation of HUVEC with bFGF is associated with a transient increase in steady-state DAF mRNA and de novo protein synthesis.


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Fig. 5.   Increased steady-state DAF mRNA and de novo protein synthesis in bFGF-induced DAF expression. A and B: HUVEC were pretreated with cycloheximide (CHX; 3 µg/ml) or vehicle alone for 30 min before the addition of bFGF (20 ng/ml) and culture for up to 24 h. A: total RNA was isolated, and Northern blots were prepared. Lane 1, unstimulated EC; lane 2, bFGF 3 h; lane 3, bFGF 6 h; lane 4, bFGF 12 h; lane 5, bFGF 24 h; lane 6, CHX alone; lane 7, bFGF 6 h + CHX; lane 8, bFGF 12 h + CHX. B: levels of mRNA were quantified with densitometric scanning. Values were corrected with respect to ethidium bromide-stained rRNA loading patterns, and an arbitrary value of 1 was assigned to unstimulated EC. C: HUVEC were pretreated with CHX (3 µg/ml) or vehicle alone for 30 min before the addition of bFGF (20 ng/ml) and culture for a further 48 h. After harvesting, DAF expression was measured by flow cytometry with MAb 1H4. Data are expressed as RFI ± SD (n = 3). The figure is representative of 3 similar experiments performed on separate EC lines. *P < 0.001.

Effect on DAF expression of combined stimulation of EC with bFGF and VEGF. bFGF and VEGF have been found to exert synergistic effects on EC proliferation and microtubule and neovessel formation both in vitro and in vivo (1, 15). As shown in Fig. 6, treatment of EC for 48 h with both bFGF and VEGF led to a significant increase in DAF expression above that seen with either growth factor alone (RFI 86 ± 9.8) and this exceeded the value calculated for an additive effect (RFI 64 ± 12; P < 0.01). These data suggest that costimulation of the discrete signaling pathways used by the two growth factors, as may occur at sites of angiogenesis, results in at least an additive and most likely a synergistic effect on DAF expression.


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Fig. 6.   Effect of costimulation of HUVEC with bFGF and VEGF on DAF expression. Monolayers of EC were stimulated for 48 h with bFGF (20 ng/ml), VEGF (25 ng/ml), or a combination of both. After harvesting, DAF expression was measured by flow cytometry with MAb 1H4. DAF expression is presented as RFI ± SD (n = 3). RFI for bFGF and VEGF (RFI ± SD = 86 ± 9.8) exceeds calculated value for an additive effect (64 ± 12) with the chi 2-test. The figure is representative of 4 similar experiments. *P < 0.01.

Effect of neutralizing antibodies on bFGF- and VEGF-induced DAF expression. Previous studies suggested that complex interactions exist between bFGF and VEGF in terms of their effects on EC function (23, 39). We therefore performed antibody crossover studies with neutralizing antibodies against bFGF and VEGF to assess the potential influence of each growth factor on the ability of the other to induce DAF upregulation. As shown in Fig. 7, the anti-bFGF antibody inhibited bFGF-induced DAF but had no effect on VEGF-induced DAF and likewise the anti-VEGF antibody inhibited VEGF but not bFGF-induced upregulation of DAF. Hence, it appears that bFGF and VEGF act independently in terms of changes in DAF expression. The lower fluorescence intensities observed reflect the use of a different directly FITC-labeled anti-DAF MAb.


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Fig. 7.   Effect of anti-bFGF and anti-VEGF Abs on growth factor-induced DAF expression. A: HUVEC were stimulated for 48 h with bFGF (20 ng/ml) or VEGF (25 ng/ml) in the presence or absence of either anti-bFGF or anti-VEGF Abs (50 µg/ml). After harvesting, single-cell suspensions were analyzed by flow cytometry for the expression of DAF with FITC-labeled anti-DAF MAb 67. DAF expression is presented as RFI ± SD (n = 3). The figure is representative of 3 similar experiments performed on different EC cultures.

Effect of bFGF-induced DAF expression on endothelial resistance to complement-mediated injury. The binding of complement factor C3 to the EC surface was used to investigate whether bFGF-induced DAF could afford increased protection to EC against complement-mediated injury. Unstimulated and growth factor-treated HUVEC were opsonized with RMAC8, an IgG2a anti-endoglin MAb. IgG2a is the optimal murine isotype for complement fixation, and endoglin is highly expressed on the EC surface and was not influenced by incubation of HUVEC with growth factors for 48 h (endoglin RFI: unstimulated EC 82 ± 2.5, bFGF-treated EC 86.8 ± 5.6, and bFGF + VEGF-treated EC 81.8 ± 4.9). After opsonization, HUVEC were incubated with 20% NHS for 3 h, after which C3 binding to the cell surface was quantified by flow cytometry with a FITC-labeled antibody against C3. Stimulation of EC for 48 h with bFGF led to a reduction in C3 deposition on the cell surface by up to 60% compared with unstimulated cells (P < 0.01; Fig. 8A). Furthermore, addition of 10 mM EDTA to the NHS completely inhibited C3 binding, consistent with the response being dependent on complement activation (data not shown). To confirm the role of DAF in the reduction of C3 binding observed, the anti-DAF MAb 1H4, which inhibits DAF function but does not fix complement, was included in the assay. In addition, we also studied the inhibitory MAb (A35) against CD59, which would not be expected to inhibit C3 binding. As shown in Fig. 8B, the addition of MAb 1H4, but not MAb A35, markedly increased the binding of C3 to unstimulated, opsonized EC exposed to 20% NHS. Moreover, the reduction in C3 binding seen in response to bFGF treatment was reversed by the presence of MAb 1H4, with levels of C3 deposited on the cell surface becoming equivalent to those observed on unstimulated EC in the presence of MAb 1H4 (Fig. 8B). The cytoprotective effects of bFGF and VEGF combined were greater than that of bFGF alone, with a further significant fall in C3 binding observed (P < 0.05; Fig. 8C).


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Fig. 8.   Functional effects of bFGF-induced increase in EC DAF expression. A-C: EC were cultured overnight at 37°C before stimulation for 48 h with bFGF (20 ng/ml) alone or bFGF (20 ng/ml) + VEGF (25 ng/ml). After harvesting, EC were incubated with the anti-endoglin MAb RMAC8 or plain medium alone for 30 min at 4°C. The EC were then washed in Hanks' balanced salt solution-1% BSA before addition of up to 20% NHS for 3 h at 37°C. Binding of C3 was detected by flow cytometry with FITC-conjugated rabbit anti-human C3. A: % C3 binding ± SD (n = 3) to unstimulated and bFGF-stimulated HUVEC, with binding to unstimulated EC shown as 100%. The negative control represents C3 binding in the presence of heat-inactivated human serum. B: C3 binding (RFI ± SD, n = 3) on unstimulated (filled bars) and bFGF-stimulated (hatched bars) HUVEC in the presence of inhibitory MAbs 1H4 (anti-DAF) and A35 (anti-CD59). C: % C3 binding ± SD (n = 3) to unstimulated, bFGF-stimulated, and bFGF + VEGF-stimulated EC, with binding to unstimulated EC shown as 100%. D: Unstimulated (filled bar), bFGF-treated (gray bar), and bFGF + VEGF-treated EC (open bar) were loaded with calcein-acetoxymethyl ether and opsonized with RMAC-8 before exposure to baby rabbit complement for 45 min at the concentrations shown. Calcein release was measured, and % EC lysis was calculated. The figure is representative of 3 similar experiments performed on separate HUVEC cultures. *P < 0.05; **P < 0.01.

To assess the physiological relevance of the reduction in C3 binding observed, HUVEC were loaded with calcein-AM, opsonized with MAb RMAC8, and exposed to baby rabbit complement. Endothelial lysis was subsequently measured by estimation of calcein release. As shown in Fig. 8D, pretreatment of EC with bFGF alone for 48 h was cytoprotective and significantly reduced cell lysis after exposure to 5% and 7.5% complement. Furthermore, the additional fall in C3 binding seen when bFGF and VEGF were combined (Fig. 8C) was translated into a marked reduction in complement-mediated cell lysis of >50% compared with that seen with bFGF alone (Fig. 8D). These observations indicate that the increased levels of cell surface DAF seen in response to bFGF provide additional protection to EC against complement-mediated injury and that this is significantly enhanced by the presence of VEGF.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although primarily considered as mitogens, bFGF and VEGF exert additional effects on the vascular endothelium, including vasodilation, increased vascular permeability, upregulation of VEGF receptors, release of proteolytic enzymes, and expression of antiapoptotic genes (5, 10). The actions of bFGF and VEGF are complementary and when present together may result in a synergistic effect on angiogenesis (1, 15, 36). These potent proangiogenic effects have been used therapeutically to promote new vessel growth for the prevention of ischemic events in peripheral vascular and coronary artery disease (17, 50), and recent studies demonstrated that both bFGF and VEGF may induce the development of neovessels and remodel preexisting collaterals (14, 16).

The endothelium is continuously exposed to activated complement, particularly during inflammation (30, 38). We showed previously (26) that the proinflammatory mediators TNF-alpha , IFN-gamma , and MAC can induce DAF expression through a PKC-independent pathway, so enhancing cytoprotection against activated complement. In addition, we also described (20, 24) a distinct PKC-dependent pathway for DAF induction utilized by VEGF and thrombin. We now provide evidence that bFGF and VEGF upregulate DAF expression on EC by distinct but complementary signaling pathways, which exert a synergistic effect when combined. These data suggest that during angiogenesis bFGF and VEGF may exert a beneficial influence above and beyond induction of neovessel formation, namely, enhanced cytoprotection against complement-mediated vascular injury.

After treatment with bFGF, endothelial DAF was increased by up to fourfold. This upregulation of DAF was dependent on de novo protein synthesis and followed an increase in steady-state mRNA. Changes in DAF mRNA in response to bFGF were indirect and presumably require synthesis of an intermediate protein(s). This is in contrast with the direct effects of thrombin and TNF-alpha , which increase DAF mRNA in the presence of CHX, but in keeping with the effect observed for VEGF (20). The relatively delayed time course of DAF protein expression is comparable to that seen in response to VEGF (26) and in the regulation of some other EC activation antigens induced via transactivation, such as Thy-1 (27). Moreover, the prolonged expression of DAF suggests that in vivo DAF expression will be persistent rather than transient in the face of chronic exposure to bFGF and VEGF, such as during angiogenesis. The concentrations of VEGF and bFGF required to alter EC function in this, and other, in vitro studies (36) exceed those reported in human serum. However, this may reflect the fact that higher concentrations are achieved locally at sites of inflammation and/or angiogenesis.

Although the induction of DAF by bFGF was PKC independent, that induced by VEGF was completely abrogated by inhibition of PKC. Comparison of the effects of the two growth factors on EC proliferation showed that bFGF was a more potent mitogen than VEGF and that VEGF-induced EC proliferation was completely inhibited by PKC antagonists. In contrast, although bFGF-induced proliferation was reduced by inhibition of PKC, a significant proportion of the response was PKC independent, as previously observed in vivo (12). Further investigation revealed additional differences, with bFGF-induced DAF upregulation dependent on activation of p38 and p44/42 MAPK, whereas the response to VEGF is regulated by p38 but not p44/42 (24). Although the precise role of MAPK in bFGF-mediated EC functions remains unclear, it has been shown that bFGF can activate both p38 and p44/42 MAPK in EC (44). Furthermore, the interaction of these pathways is likely to be important, with a recent study demonstrating regulatory cross talk (43). However, endothelial MAPK signaling pathways remain to be fully elucidated and caution is required in interpreting results obtained with pharmacological inhibitors (3).

Previous studies demonstrated interdependence of bFGF and VEGF so that in certain circumstances VEGF expression in EC can be modulated by bFGF (39), and another study reported that VEGF-induced angiogenesis is dependent on endogenous bFGF (23). These data suggest that complex interactions exist between these two growth factors, which may result in synergy (1, 15, 39). Using neutralizing antibodies we found that, in terms of DAF expression, bFGF and VEGF act independently so that the effects of each were blocked only by its specific neutralizing antibody. Furthermore, experiments in which bFGF- or VEGF-conditioned EC supernatants were transferred to unstimulated cells (not shown) suggested that the intermediate protein(s) required for an increase in DAF steady-state mRNA are not secreted and presumably therefore act intracellularly.

To assess the functional role of growth factor-induced DAF we measured C3 binding to the surface of opsonized EC after complement activation. Pretreatment with bFGF resulted in a 60% reduction of C3 binding. Furthermore, when EC were treated with both bFGF and VEGF a further reduction in C3 binding was seen. Therefore, because the activation of C5 is critically dependent on an absolute excess of activated C3 (4), the reduction in C3 binding observed is especially relevant. Thus the reduced C3 binding was translated into a significant reduction in complement-mediated EC lysis, an effect that was maximal in EC exposed to a combination of bFGF and VEGF.

The complement system has been implicated in the pathogenesis of atherosclerosis, myocardial infarction, and the accelerated atherosclerosis of transplantation (9, 32, 37, 46). Complement components from C1q to C9 have been identified in atherosclerotic lesions and are expressed at significantly higher levels than in lesion-free areas of the same artery (53). Moreover, there is clear evidence for ongoing complement activation, with C3d, C4d, and C5b-C9 all being present (48, 53). Evidence to date implicates a number of potential mechanisms for local complement activation within the arterial wall. These include deposition of immune complexes such as those generated by autoantibodies against oxidized lipoproteins (34), activation by cholesterol crystals (42), C-reactive protein (53), or modified low-density lipoprotein (47). Furthermore, the generation of C5b-9 in sublytic amounts may result in cellular activation and adhesion molecule upregulation, chemokine secretion, growth factor release, and smooth muscle cell and EC proliferation (reviewed in Ref. 45). Although complement-inhibitory proteins have also been identified in atherosclerotic lesions, their expression, regulation, and function are less well understood (40, 41). However, it has been suggested that the expression of DAF may help reduce complement-mediated injury in atherosclerosis (33). Hence, the upregulation of DAF on EC described herein may represent an important mechanism for enhanced vascular cytoprotection with subsequent slowing of the progression of atherosclerotic lesions. This cytoprotective effect may also be of benefit during therapeutic angiogenesis, both as a means by which the neovasculature is protected against complement-mediated injury and also by minimizing local complement activation at sites of atherosclerosis. To this end, it is of interest to note that bFGF has been reported to have a cytoprotective effect in a rat ischemia-reperfusion model (35).

It is now accepted that bFGF and VEGF act as survival factors for the developing neovasculature during angiogenesis, predominantly through their activation of antiapoptotic genes including Bcl-2 and A1 (13, 19, 54). Our study demonstrates an additional cytoprotective function of these growth factors via the upregulation of DAF. An improved understanding of the mechanisms of vascular cytoprotection, including those relating to the control of complement activation, will facilitate the development of novel therapeutic approaches by which endothelium can be conditioned for the prevention and treatment of vascular inflammatory diseases including atherosclerosis and its complications.


    ACKNOWLEDGEMENTS

We thank Paul Morgan, Doug Lublin, Tony d'Apice, Teizo Fujita, and John Atkinson for the generous provision of reagents used in this study and the staff of the surgical and maternity units of Hammersmith Hospital for help with tissue collection.


    FOOTNOTES

This study was funded by Arthritis Research Campaign Grant M0620 to J. C. Mason and in part by a discretionary professorial award from the British Heart Foundation.

Address for reprint requests and other correspondence: J. C. Mason, The BHF Cardiovascular Medicine Unit, Imperial College School of Science, Technology and Medicine, Hammersmith Hospital, Du Cane Rd., London W12 ONN, UK (E-mail: justin.mason{at}ic.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00339.2001

Received 24 July 2001; accepted in final form 31 October 2001.


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