Published online before print December 14, 2006, doi: 10.1161/01.ATV.0000255309.38699.6c
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© 2007 American Heart Association, Inc.
Vascular Biology |
Vascular Endothelial Growth Factor Synergistically Enhances Induction of E-Selectin by Tumor Necrosis Factor-
From BHF Laboratories, Department of Medicine, University College London, United Kingdom.
Correspondence to Ian Zachary, BHF Laboratories, Department of Medicine, The Rayne Building, University College London, 5 University Street, London WC1E 6JJ, United Kingdom. E-mail i.zachary{at}ucl.ac.uk
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Abstract |
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Objective— The regulation of endothelial cell adhesion molecules (CAMs) by vascular endothelial growth factor (VEGF) was investigated in cell cultures and in a rabbit model of atherogenic neointima formation.
Methods and Results— VEGF regulation of vascular CAM-1 (vascular cell adhesion molecule), intercellular CAM-1 (intercellular adhesion molecule), and E-selectin were investigated in human umbilical vein endothelial cells using quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and flow cytometry, and in the rabbit collar model of atherogenic macrophage accumulation by immunostaining. VEGF alone caused no significant induction of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, or E-selectin compared with tumor necrosis factor-
. In both hypercholesterolemic and normal rabbits, adenoviral VEGF-A165 expression caused no increase in endothelial vascular cell adhesion molecule-1 or E-selectin. In contrast, pretreatment of human umbilical vein endothelial cells with VEGF significantly increased E-selectin expression induced by tumor necrosis factor-, compared with tumor necrosis factor- alone, whereas vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 were unaffected. VEGF similarly enhanced IL-1ß–induced E-selectin upregulation. VEGF also synergistically increased tumor necrosis factor-–induced E-selectin mRNA and shedding of soluble E-selectin. Synergistic upregulation of E-selectin expression by VEGF was mediated via VEGF receptor-2 and calcineurin signaling.
Conclusions— VEGF alone does not activate endothelium to induce CAM expression; instead, VEGF "primes" endothelial cells, sensitizing them to cytokines leading to heightened selective pro-inflammatory responses, including upregulation of E-selectin.
VEGF alone caused no significant induction of cell adhesion molecule expression in endothelial cells or during neointima formation in vivo. However, VEGF pretreatment enhanced E-selectin expression induced by TNF-. These results indicate that VEGF "primes" endothelial cells to enhance selective responses to proinflammatory cytokines
Key Words: cell adhesion molecules • endothelial cells • IL-1ß
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Introduction |
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The endothelium has several important functions that include providing a nonadhesive, nonthrombotic barrier between the blood and the underlying tissues. In atherosclerosis, or in response to injury or inflammatory cytokines such as tumor necrosis factor-
(TNF-), the endothelium becomes activated and cell adhesion molecules (CAMs) are rapidly induced.1,2 In particular, members of the immunoglobulin superfamily of CAMs, such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as the selectin family members, E-selectin and P-selectin, have crucial roles in the adhesion and migration of monocyte/macrophage infiltration into atherosclerotic lesions during the early and subsequent stages of atherosclerosis in a variety of animal models.1–7
Recent findings suggest that the angiogenic cytokine, vascular endothelial growth factor (VEGF or VEGF-A) may act as a proinflammatory cytokine by increasing ICAM-1, VCAM-1, and E-selectin mRNA in cultured endothelial cells through activation of nuclear factor-
B (NF-B),8,9 an important transcription factor mediating CAM gene expression. So far, however, the relevance of VEGF-induced surface CAM expression for endothelial function has not been clarified. Given that VEGF is both a major focus of interest as a therapeutic angiogenic cytokine for coronary artery disease, and, paradoxically, has been reported to enhance atherosclerotic lesion formation,10–13 elucidation of the effects of VEGF on endothelial CAM expression has important practical, as well as biological, implications.
VEGF elicits an array of biological effects on endothelial cells in vivo and in vitro including survival, proliferation and migration, nitric oxide and prostacyclin (PGI2) production, and increased vascular permeability.14,15 VEGF exerts its actions by binding two cell surface protein kinase receptors, VEGF receptor (VEGFR)2/KDR/Flk-1 and VEGFR1/Flt-1. Most biological responses triggered by VEGF in endothelial cells are mediated primarily by VEGFR2 and activation of multiple early signaling cascades, including phosphatidylinositol 3'-kinase (PI3K)-dependent Akt/PKB, phospholipase C-
, mitogen-activated protein/extracellular signal-regulated kinase and calcineurin/nuclear factor of activated T-cells pathways.13–17
In the present study, we investigated the ability of VEGF to stimulate CAM expression in human umbilical vein endothelial cells (HUVECs) and in vivo. VEGF induced minor changes in CAM mRNA, which were insufficient to increase CAM expression at the cell surface as determined by sensitive flow cytometry, although TNF- strongly induced CAM mRNA and cell surface expression. Adenoviral overexpression of VEGF also had no effect on endothelial VCAM-1 and E-selectin expression in a rabbit model of neointima formation and neointimal macrophage accumulation. Surprisingly, when HUVECs were pre-incubated with VEGF before TNF- treatment, E-selectin mRNA and cell surface expression were synergistically increased as compared with TNF- alone, whereas other CAMs were unaffected. VEGF also synergistically enhanced TNF-–induced shedding of soluble E-selectin. These findings demonstrate that VEGF is not able to stimulate endothelial CAM expression alone, but interacts with TNF- to synergistically and selectively enhance E-selectin expression.
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Experimental Procedures |
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Materials and Methods
For sources of cytokines, antibodies, and other reagents see supplemental experimental procedures (http://atvb.ahajournals.org).
Cell Culture
HUVECs were cultured as described online in the supplement.17
Collar Placement and Adenoviral Gene Transfer
Animal experiments were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 and the Animal Care and Ethics Guidelines of University College London, UK. Maintenance of New Zealand White rabbits on a high-cholesterol diet, collar placement around the carotid artery, and delivery of adenoviruses (Ark Therapeutics Ltd, Kuopio, Finland) encoding VEGF-A165 (Ad.VEGF-A165) or LacZ (Ad.LacZ) were performed as described.11
RNA Isolation, Reverse-Transcription Polymerase Chain Reaction and Quantitative Real-Time Polymerase Chain Reaction
Total RNA isolation from rabbit carotid arteries and HUVECs, reverse-transcription polymerase chain reaction, and quantitative real-time polymerase chain reaction were performed as described (supplemental Table I, available online at http://atvb.ahajournals.org).11,17,18
LacZ Staining and Immunohistochemistry
Detection of ß-galactosidase and immunostaining of VCAM-1, E-selectin, macrophages, CD31, and VEGF were performed as described.11
Morphometry and Image Analysis
Intima/media ratios, and staining of CD31 (endothelial neovascularization), RAM-11 (macrophages), VCAM-1, and E-selectin in collared rabbit carotid arteries were quantified as previously described using Image J.11,18
Enzyme-Linked Immunosorbent Assay
Analysis of total (cell surface and intracellular) VCAM-1 expression was performed as described.19 E-selectin, ICAM-1, and von Willebrand factor were analyzed by substituting anti-VCAM-1 with anti-E-selectin (5 µg IgG1/mL), anti-ICAM-1 (1 µg IgG1/mL), or anti- von Willebrand factor (1.2 µg IgG1/mL).
Flow Cytometry
Cell surface VCAM-1, E-selectin, ICAM-1, and PECAM-1 was measured in confluent HUVECs as described.19
Soluble E-selectin Immunoassay
Human soluble E-selectin (sE-selectin) was measured by commercial immunoassay in HUVEC culture supernatants.
Statistical Analysis
Differences between different treatment groups in rabbits were evaluated by ANOVA and Bon Ferroni correction (SPSS). Statistical analysis of cell culture experiments was performed using Student t test or 1-way or 2-way ANOVA with post hoc analysis by Fisher PLSD, when appropriate. Results are shown as means±SE or mean±SD, and P<0.05 was considered significant.
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Results |
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VEGF Does Not Upregulate Endothelial Protein Expression of E-selectin, ICAM-1, or VCAM-1
The effects of VEGF on CAM mRNA levels were measured in HUVECs by quantitative real-time reverse-transcription polymerase chain reaction and normalized to GAPDH expression (Figure 1A). Tissue factor (TF) mRNA, previously shown to be markedly upregulated by VEGF,17 was increased
70-fold 90 minutes after addition of VEGF and returned to near control levels by 24 hours. VEGF increased E-selectin, VCAM-1, and ICAM-1 mRNA expression to maximum levels of, respectively, 7-fold after 45 minutes, 29-fold after 3 hours, and 2-fold after 1.5 hours (Figure 1A). Levels of PECAM-1 (CD31) mRNA, constitutively expressed by HUVECs, were unchanged by VEGF incubation (data not shown). To assess the biological relevance of VEGF-induced increases in CAM expression, we compared the effects of VEGF with those of the inflammatory cytokine, TNF-. The results of this comparison showed that the modest inductions of CAMs by VEGF were negligible compared with the strong induction of E-selectin, ICAM-1, and VCAM-1 by TNF- alone: 900-, 300-, and 800-fold increases in mRNA, respectively (Figure 1B).
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, yellow) and E-selectin (•, red) mRNA levels were modestly increased by 3 hours after addition of VEGF, whereas ICAM-1 (
, green) mRNA levels were virtually unchanged. TF mRNA levels increased as expected (
, blue). Reverse-transcription polymerase chain reaction results are expressed as fold-increases ±SD above the control level (in basal unstimulated cells) normalized to GAPDH expression. B, Levels of VCAM-1 (open bars), ICAM-1 (gray bars), and E-selectin (black bars) mRNA were measured in HUVECs treated for 1.5 hours (ICAM-1 and E-selectin) or 3 hours (VCAM-1) with 25 ng/mL VEGF-A (V), or for 6 hours with 100 U/mL TNF-
Because VEGF has previously been reported to stimulate protein expression of CAMs in endothelial cells,8,9 we examined the possibility that VEGF might increase CAM expression despite the relatively small effect of VEGF on mRNA induction compared with TNF-. Although TNF- treatment induced increases in E-selectin (Figure 1C), ICAM-1 (Figure 1D), and VCAM-1 (Figure 1E) of 6-fold, 5.6-fold, and 2-fold above basal unstimulated levels, respectively, VEGF failed to induce any significant increase in CAM expression after any of the incubation times tested (Figure 1C through 1E). In each experiment, von Willebrand Factor, which is known to be increased by VEGF,20 was significantly increased after 24-hour incubation with VEGF by 44.3±16.3% (P<0.05, n=3, results not shown). Measurement of cell surface CAMs using flow cytometry confirmed the results of enzyme-linked immunosorbent assays, and was unable to detected a subpopulation of cells with increased CAM expression in response to VEGF; fluorescence-activated-cell sorter profiles provided clear evidence that CAM expression on the cell surface was not increased by VEGF, whereas TNF- strongly upregulated CAM expression (data not shown).
Effect of VEGF on VCAM-1 and E-selectin Expression in a Model of Atherogenic Macrophage Accumulation
Because of the key role thought to be played by upregulation of endothelial VCAM-1 in neointimal macrophage infiltration during the early stages of atherosclerosis, the effects of high-efficiency adenoviral VEGF-A165 (Ad.VEGF) expression on VCAM-1 expression were investigated in a model of neointimal thickening and neointimal macrophage accumulation induced by placement of an inert silastic collar around the carotid artery of rabbits fed a high-cholesterol diet. Staining for ß-galactosidase in arteries transduced with Ad.LacZ revealed abundant strongly stained cells in the adventitia consistent with a high efficiency of gene transfer (5% to 10%) (supplemental Figure IA).
Expression of the VEGF transgene after periadventitial delivery of Ad.VEGF to carotid arteries was confirmed by reverse-transcription polymerase chain reaction (supplemental Figure IB). No VEGF transgene expression was detected in Ad.LacZ-transduced arteries, in segments of the transduced carotid arteries distal to the collared arterial region, in contralateral noncollared control arteries, or in nontargeted tissues (results not shown), indicating that the perivascular collar localized transgene expression to the collared region of the artery. Immunostaining of sections of transduced arteries with a specific antibody to VEGF showed strong expression of VEGF in the adventitia of Ad.VEGF-transduced arteries (supplemental Figure IC).
Consistent with previous findings,11 immunostaining with macrophage-specific RAM11 and specific VCAM-1 antibodies showed that neither neointimal macrophage accumulation nor endothelial VCAM-1 expression occurred in the collared arteries of rabbits fed a normal diet. Ad.VEGF delivery to collared arteries in rabbits on a normal diet significantly increased neointima formation and adventitial neovascularization, but had no effect on either neointimal macrophage accumulation or VCAM-1 expression (supplemental Figure ID; results not shown). However, as compared with Ad.lacZ, Ad.VEGF delivery had no significant neointima-increasing effect in the collared arteries of cholesterol-fed rabbits (Figure 2A). Collar placement in hypercholesterolemic rabbits induced lesions containing RAM11-positive macrophages and marked endothelial VCAM-1 and E-selectin expression (Figure 2B and 2C), but Ad.VEGF caused no significant increase in either neointimal macrophage accumulation (Figure 2B) or endothelial VCAM-1 or E-selectin (Figure 2C), although Ad.VEGF did significantly increase adventitial angiogenesis (supplemental Figure II).
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VEGF and TNF- Synergize to Upregulate E-Selectin Expression
We next investigated the possibility that VEGF could modulate CAM expression induced by TNF-. VEGF pretreatment of HUVECs for 24 hours before TNF- stimulation for the last 6 hours of incubation increased E-selectin expression by 61.7±5.8% from the level induced by TNF- alone (Figure 3A; P=0.004, n=3). In contrast, TNF-–induced ICAM-1 expression was not enhanced with VEGF pretreatment (Figure 3A).
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Enzyme-linked immunosorbent assay data were confirmed by flow cytometry. Figure 3B shows a representative 2-dimensional fluorescence-activated cell sorter overlay histogram for cell surface E-selectin expression in HUVECs. The fluorescein isothiocyanate fluorescence profile showed that basal E-selectin expression in unstimulated cells was negligible (Figure 3B), similar to the profile of cells incubated with VEGF alone or to isotype-treated controls (data not shown). TNF- treatment upregulated cell surface E-selectin, whereas VEGF pretreatment before TNF- incubation further increased cell surface E-selectin expression by 77.2±15.0% (Figure 3C, P<0.005, n=3). In parallel, VEGF preincubation did not significantly increase either ICAM-1 or VCAM-1 above TNF-–induced levels (Figure 3C). Constitutive levels of PECAM-1 (ie, without TNF- stimulation) were not altered significantly by VEGF (data not shown).
Synergistic upregulation of E-selectin expression by VEGF and TNF- also occurred at 10 U/mL (0.5 ng/mL) TNF- (Figure 3D). Indeed, pretreatment with VEGF before addition of 10 U/mL TNF- induced a greater synergistic effect than 100 U/mL TNF- (143% versus 71%, respectively, above the corresponding TNF- controls), as shown by enzyme-linked immunosorbent assay.
IL-1ß and TNF- stimulated E-selectin protein expression to a similar extent, 25-fold above the level in control unstimulated cells (data not shown). IL-1ß synergized with VEGF to enhance E-selectin expression (48.2±10.5% increase from level of IL-1ß control, P=0.005, n=3; Figure 3E), although in parallel cell cultures, TNF- caused a greater synergistic effect with VEGF than IL-1ß (77.2±15.0% enhancement).
VEGF and TNF- Synergize to Upregulate E-Selectin mRNA Levels
Quantitative real-time reverse-transcription polymerase chain reaction showed that VEGF and TNF- synergize to increase E-selectin transcription (Figure 3F). The induction in E-selectin mRNA increased from 830-fold above the control unstimulated level with TNF- alone to 1500-fold with VEGF and TNF- (81% increase, P
0.05, n=3). Neither ICAM-1 nor VCAM-1 mRNAs were significantly enhanced by the combination of VEGF and TNF- above the levels induced by TNF- alone (data not shown). Consistent with previous findings,21,22 VEGF and TNF- synergized to upregulate TF mRNA expression (P<0.05), whereas VEGF/TNF- caused no synergistic upregulation of COX-2 (supplemental Figure III). Although VEGF induces rapid upregulation of COX-2, COX-2 expression declines to basal levels after 6 hours and is not detectable after 24 hours.16,17
VEGF/TNF- Synergy Requires Preincubation With VEGF
HUVECs were preincubated with VEGF for various times (0 to 24 hours) before TNF- stimulation and then analyzed for E-selectin expression by flow cytometry to find the minimum preincubation time needed with VEGF for synergy to occur. The maximum synergistic effect of VEGF/TNF- on E-selectin required at least 4 hours pretreatment with VEGF (Figure 4A).
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The possibility that VEGF might enhance E-selectin expression via secretion of a soluble factor was tested by transferring media from VEGF-treated cells to another set of cultures that were then immediately incubated with TNF- for 6 hours. The results showed that media from VEGF-treated cells did not enhance E-selectin expression (Figure 4B), suggesting that VEGF does not induce production of a diffusible secreted factor that mediates synergy with TNF-. In the wells where the VEGF-containing media had been removed after incubation, addition of fresh media without VEGF but containing TNF- still caused synergy (Figure 4B). These experiments confirmed that pre-incubation with VEGF is needed for synergy with TNF-, although VEGF does not need to be present when cells are stimulated with TNF-.
VEGF/TNF- Synergy Is Mediated by VEGFR2 and the Calcineurin/Nuclear Factor of Activated T-Cells Signaling Pathway
We examined the receptor and second messenger mechanisms mediating the VEGF/TNF- synergistic effect on E-selectin. Placental growth factor (PlGF), a ligand for VEGFR1 but not for VEGFR2, failed to synergize with TNF- to enhance E-selectin expression, and PlGF had no effect on the VEGF/TNF- synergy when HUVECs were preincubated with a combination of PlGF and VEGF (Figure 5A), as shown by flow cytometry. Preincubation with the specific VEGFR2 inhibitor, SU5614, almost completely blocked the synergistic effect on E-selectin expression (84.6±8.1% inhibition, P=0.005), indicating that VEGFR2 mediated this effect (Figure 5B).
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Cyclosporin A (0.2 µmol/L), an inhibitor of the protein phosphatase activity of calcineurin/protein phosphatase 2B, partially blocked the VEGF/TNF- synergism implicating the involvement of the calcineurin/nuclear factor of activated T-cells signaling pathway (59.3±0.8% inhibition P=0.03, n=3; Figure 5C). Inhibition of either the extracellular signal-regulated kinase 1/2 pathway using the specific MAP kinase kinase (mitogen-activated protein/extracellular signal regulated kinase) inhibitor, U0126 (10 µmol/L), or PI3K-dependent Akt activation using LY294002, did not significantly inhibit the synergistic cooperation between VEGF and TNF- (Figure 5D and results not shown).
VEGF/TNF- Synergism Increases E-Selectin Shedding
After endothelial activation, surface E-selectin undergoes shedding to release soluble isoforms.23 To test the effect of VEGF on TNF-–induced shedding, culture media were removed after VEGF pretreatment and subsequent incubation with TNF- for 6 hours and replaced with fresh media not containing VEGF or TNF-. Soluble E-selectin (sE-selectin) shed into the media was measured using a sensitive sandwich enzyme-linked immunosorbent assay (Figure 6). TNF-–induced sE-selectin expression in HUVECs was detectable after 2 hours and increased up to 24 hours (Figure 6). While VEGF alone did not cause any detectable increase in the level of sE-selectin produced by HUVECs, VEGF preincubation synergistically increased TNF-–induced shedding of sE-selectin at all time points, an effect that was statistically significant (P<0.05) after 6, 20, and 24 hours (Figure 6).
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Discussion |
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The role of VEGF in atherosclerosis is contentious because studies have reached differing conclusions as to whether VEGF can either inhibit or promote intimal thickening and atherosclerotic lesion formation in a variety of animal models. A proatherogenic role of VEGF is supported by reports that VEGF upregulates expression of inflammatory CAMs in endothelial cells, including VCAM-1, ICAM-1, and E-selectin.8,9 Upregulation of endothelial CAMs, particularly VCAM-1, is recognized to play a key role in the activation of endothelium in inflammation, and a key mediator of monocyte/macrophage adhesion and infiltration in early and later atherosclerotic lesions responsible for the excessive accumulation of macrophages in the atherosclerotic plaque. Whether or how VEGF regulates endothelial CAM expression is therefore a question of central importance for the pathophysiology of this cytokine, which also has ramifications for the therapeutic potential of VEGF in ischemic heart disease.
The first major finding of this article is that VEGF alone is unable to induce protein expression of endothelial VCAM-1, ICAM-1, and E-selectin in endothelial cells expressing high levels of VEGFR2/KDR. In addition, adenoviral overexpression of VEGF in a model of atherogenic neointima formation and macrophage accumulation also had no significant effect on endothelial VCAM-1 and E-selectin expression in vivo. Our previous findings showed that liposome-mediated plasmid VEGF gene delivery to the collared arteries of hypercholesterolemic rabbits, reduced neointima formation, neointimal macrophage accumulation, and endothelial VCAM-1 expression,11 but this study did not examine the effects of high-efficiency adenoviral VEGF gene delivery. Importantly, neither low- nor high-efficiency VEGF expression caused any increase in VCAM-1 expression. The different effects of plasmid and adenoviral VEGF on intimal thickening, macrophage accumulation, and VCAM-1 expression in the collar model may be attributable to an arterioprotective effect of low intra-arterial VEGF concentrations that is impaired at the higher local concentrations of VEGF produced by adenoviral expression.11 Interestingly, Ad.PlGF gene delivery in the same model in parallel with the study of Ad.VEGF presented here, increased neointimal thickening, macrophage accumulation and endothelial VCAM-1 expression in collared arteries in hypercholesterolemic rabbits.24 The present results therefore indicate a striking contrast in the biological effects of VEGF and PlGF in atherogenic lesion formation in rabbits.
Whereas VEGF significantly increased VCAM-1, ICAM-1, and E-selectin mRNA expression in endothelial cells, these effects were extremely small compared with the effect of TNF-. Kim et al reported that VEGF increased VCAM-1, ICAM-1, and E-selectin protein expression, as determined by Western blot.8,9 In contrast, we found that VEGF had no significant effect on CAM protein expression as judged by sensitive and quantitative enzyme-linked immunosorbent assays of total cellular CAM expression or measurement of cell surface CAM expression by flow cytometry. The lack of CAM upregulation as determined by flow cytometry also precluded the possibility that there was a subpopulation of cells that could upregulate CAM expression in response to VEGF. Previous studies examined effects of VEGF in freshly isolated HUVECs cultured in M199 medium with serum,8,9 whereas our studies used commercially available HUVECs cultured in supplemented EBM. However, we conclude that differences in the source and culture of HUVECs are unlikely to account for our failure to observe an effect of VEGF alone on CAM expression. TNF- and IL-1ß strongly upregulated CAM expression at protein and mRNA level indicating that the cellular mechanisms mediating transcriptional upregulation of CAMs are not impaired in commercial HUVECs. We show here and in previous studies that VEGF strongly induces an array of signaling and cellular responses in commercially obtained HUVECs, including upregulation of multiple genes.17,25 Furthermore, we have confirmed that several key signaling and biological responses to VEGF are preserved in low-passage number freshly isolated and in commercial HUVECs.20,25 Moreover, our previous findings together with the results of adenoviral VEGF overexpression presented here also indicate that VEGF is unable to significantly enhance endothelial VCAM-1 and E-selectin expression in vivo, even under inflammatory hypercholesterolemic conditions favoring CAM upregulation. Zhang and Issekutz also showed that VEGF, in contrast to TNF-, had no effect on ICAM-1, VCAM-1, and E-selectin expression in HUVECs as determined by enzyme-linked immunosorbent assay, although this study examined effects of a 3-day treatment.26 We provisionally conclude that differences between our findings and those of other researchers may be attributable to the different approaches used to measure CAM expression, although more work is needed to establish more precisely the reasons for these differences.
The second major finding of this paper is that whereas VEGF alone had no significant effect on CAM expression, it synergistically enhanced the induction of E-selectin mRNA and cell surface protein expression by TNF-. This was a selective synergistic interaction because VEGF did not enhance TNF-–induced expression of VCAM-1 or ICAM-1. VEGF also synergized with TNF- to enhance shedding of the soluble extracellular domain of E-selectin. VEGF and TNF- have previously been shown to synergistically increase functional TF expression in endothelial cells, but it has been unclear whether cooperative interactions between these 2 cytokines are relevant for other endothelial responses. Synergistic enhancement of E-selectin expression by VEGF was mediated by VEGFR2/KDR; in addition, the inhibition of the effect by cyclosporin A, a specific inhibitor of calcineurin, indicates that a calcineurin-dependent pathway also plays a major role in this response. It has been reported that mitogen-activated protein/extracellular signal-regulated kinase/extracellular signal-regulated kinase is the convergence point of the VEGF and TNF- signaling for the synergistic upregulation of TF, which leads to activation of the transcription factor early growth response-1.27 However, inhibition of mitogen-activated protein/extracellular signal regulated kinase did not block the synergistic enhancement of E-selectin. Early growth response-1 elements have been found in some CAM promoters but are unknown for E-selectin. The nuclear factor of activated T-cells/calcineurin pathway has been implicated in expression of E-selectin28 and VEGF-induced upregulation of TF,29 and Down syndrome critical region protein 1.17 Cyclosporin A has been reported to suppress E-selectin, but not VCAM-1, induction in HUVECs by TNF-, even though the E-selectin promoter is activated by NF-B rather than nuclear factor of activated T-cells.28
E-selectin has been implicated in angiogenesis in vitro30,31 and in vivo,32 although the underlying mechanisms remain to be defined. Synergistic augmentation of TNF-–regulated E-selectin expression by VEGF might therefore be relevant for neovascularization in an inflammatory milieu, as for example in pathophysiological settings such as atherosclerosis, cancer, and rheumatoid arthritis. E-selectin is proteolytically shed by an unknown mechanism with the resulting sE-selectin, lacking transmembrane and cytoplasmic domains, being biologically active and playing a role in angiogenesis through the Src-PI3K pathway.33,34 VEGF-dependent augmentation of sE-selectin generation induced by inflammatory cytokines may contribute to the pathogenesis of cardiovascular disease, possibly through enhanced angiogenesis within atherosclerotic lesions, a phenomenon that has been hypothesized to promote intraplaque hemorrhage and destabilization leading to rupture. However, E-selectin–deficient mice develop normally and exhibited no impairment in experimentally induced angiogenesis,35 indicating that VEGF-enhanced shedding of sE-selectin is unlikely to play a key role in developmental angiogenesis, although it is not precluded that it may contribute to pathophysiological angiogenesis in disease-specific settings. The role of sE-selectin in atherosclerosis is less clear because, whereas it has proinflammatory effects on neutrophil function,36 induces monocyte chemotaxis,34 and is significantly increased in patients with coronary artery disease, the increases observed have been small and, in several studies of human populations, sE-selectin levels have not emerged as a strong predictor of cardiovascular disease.23
The importance of synergistic interactions between cytokines is becoming increasingly appreciated.37 However, this is the first report to our knowledge of enhancement of CAM expression by VEGF/TNF- synergy. Furthermore, VEGF also synergized with IL-1ß to upregulate E-selectin. Because TNF- and IL-1ß share many of the same signaling pathways, these findings suggest that VEGF-triggered signal transduction mechanisms can cooperate with a common pathway activated by inflammatory cytokines. VEGF may "prime" endothelial cells so they are capable of responding to lower levels of TNF-. Consistent with this notion, we observed relatively more synergy with a lower concentration of TNF-.
The present article is consistent with the broader conclusion that the predominant effect of VEGF alone is not proinflammatory and does not cause endothelial cell activation associated with cardiovascular disease. However, because VEGF can selectively enhance E-selectin expression induced by TNF- or IL-1ß, we propose that the overall impact of VEGF on endothelial function and, by extension, on vascular pathophysiology, may be modified by the local cytokine milieu. A fuller understanding of how VEGF interacts and synergizes with other endothelial cytokines will be key to delineating its role as both a therapeutic and pathogenic factor in cardiovascular disease.
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Acknowledgments |
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Sources of Funding
This work was supported by British Heart Foundation grants RG/02/001 and BS/94001 to I.Z.
Disclosures
None.
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Footnotes |
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Original received August 2, 2006; final version accepted November 23, 2006.
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References |
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