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
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ABSTRACT |
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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)-, interferon (IFN)-
, 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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MATERIALS AND METHODS |
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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 2-test was used. Differences
were considered significant at P values of <0.05.
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RESULTS |
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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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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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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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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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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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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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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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DISCUSSION |
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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-, IFN-
, 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-, 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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