Published online before print September 18, 2008, doi: 10.1161/CIRCRESAHA.108.184036
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© 2008 American Heart Association, Inc.
Integrative Physiology |
Isoprostanes Inhibit Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration, Tube Formation, and Cardiac Vessel Sprouting In Vitro, As Well As Angiogenesis In Vivo via Activation of the Thromboxane A2 Receptor
A Potential Link Between Oxidative Stress and Impaired Angiogenesis
From the Clinical Pharmacology Unit (R.A.B., E.S., A.G., R.T., G.K., M.D., A.S., R.H.B.), Institute of Experimental and Clinical Toxicology and Pharmacology, University Hospital Hamburg-Eppendorf; and Institute of Anatomy (S.E.), University Hospital Essen, Germany.
Correspondence to Ralf Benndorf, Clinical Pharmacology Unit, Department of Pharmacology, University Hospital Hamburg, Martinistrasse 52, Hamburg 20246, Germany. E-mail benndorf{at}uke.uni-hamburg.de
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Abstract |
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Isoprostanes are endogenously formed end products of lipid peroxidation. Furthermore, they are markers of oxidative stress and independent risk markers of coronary heart disease. In patients experiencing coronary heart disease, impaired angiogenesis may exacerbate insufficient blood supply of ischemic myocardium. We therefore hypothesized that isoprostanes may exert detrimental cardiovascular effects by inhibiting angiogenesis. We studied the effect of isoprostanes on vascular endothelial growth factor (VEGF)-induced migration and tube formation of human endothelial cells (ECs), and cardiac angiogenesis in vitro as well as on VEGF-induced angiogenesis in the chorioallantoic membrane assay in vivo. The isoprostanes 8-iso-PGF2
, 8-iso-PGE2, and 8-iso-PGA2 inhibited VEGF-induced migration, tube formation of ECs, and cardiac angiogenesis in vitro, as well as VEGF-induced angiogenesis in vivo via activation of the thromboxane A2 receptor (TBXA2R): the specific TBXA2R antagonists SQ-29548, BM 567, and ICI 192,605 but not the thromboxane A2 synthase inhibitor ozagrel blocked the effect of isoprostanes. The isoprostane 8-iso-PGA2 degraded into 2 biologically active derivatives in vitro, which also inhibited EC tube formation via the TBXA2R. Moreover, short hairpin RNA–mediated knockdown of the TBXA2R antagonized isoprostane-induced effects. In addition, Rho kinase inhibitor Y-27632 reversed the inhibitory effect of isoprostanes and the thromboxane A2 mimetic U-46619 on EC migration and tube formation. Finally, the various isoprostanes exerted a synergistic inhibitory effect on EC tube formation. We demonstrate for the first time that isoprostanes inhibit angiogenesis via activation of the TBXA2R. By this mechanism, isoprostanes may contribute directly to exacerbation of coronary heart disease and to capillary rarefaction in disease states of increased oxidative stress.
Key Words: angiogenesis • migration • isoprostanes • thromboxane A2 receptor • Rho kinase
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Introduction |
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Oxidative stress is characterized by an imbalance between increased exposure to reactive (oxygen) species, predominantly free radicals, and antioxidant defense mechanisms. Endogenously, such free radicals are generated by enzymatic sources such as the NADPH oxidase or derive from mitochondria, eg, in the pathophysiological state of tissue hypoxia.1,2 Free radicals cause direct harm to essential cellular components, such as DNA, lipids, and proteins.2 As 1 example, free radical–induced peroxidation of arachidonic acid leads to cyclooxygenase-independent in vivo formation of prostaglandin-like substances, named isoprostanes.3 Hence, endogenous formation of isoprostanes should mirror the exposure of an organism to free radicals. In line with this consideration, a growing body of evidence clearly underlines the validity of isoprostanes as in vivo markers of oxidative stress.4 Most interestingly, patients with established coronary heart disease (CHD) but also patients at high cardiovascular risk, eg, smokers, hypercholesterolemic, diabetic, and obese patients or patients with salt-sensitive or renovascular hypertension, exhibit elevated urinary or plasmatic concentrations of isoprostanes.4–11 Moreover, recently published experimental data suggest that isoprostanoids strongly accumulate in hypoxic myocardium.12 In addition, we lately identified isoprostanes as independent risk markers of CHD.5 Hence, our findings point to a more direct role isoprostanes in cardiovascular pathophysiology. Indeed, a plethora of experimental evidence demonstrate that isoprostanes are biologically active compounds.13 For instance, isoprostanes have been shown to induce vasoconstriction via the thromboxane A2 receptor (TBXA2R) and may promote the formation of atherosclerotic plaques in ApoE knockout mice via the same receptor.14,15 Taking into account the short half life of thromboxane A2 in vivo (approximately 30 seconds), isoprostanes may represent important stable ligands of the TBXA2R, especially in pathophysiological states of increased oxidative stress.16 Thus, isoprostanes may attract attention not only as valid markers of oxidative stress in vivo but also as mediators of cardiovascular pathology. In this regard, vascular remodelling has been shown to occur in the chronically hypoxic heart of patients with CHD and may be of major importance to maintain cardiac blood flow in these patients.17,18 Moreover, several groups have demonstrated that patients at high cardiovascular risk, eg, hypertensive individuals, or patients with established CHD exhibit an impaired response to angiogenic stimuli and a rarefaction of the peripheral microvascular (capillary) bed.19,20 Because recent experimental findings have indicated that synthetic TBXA2R mimetics may inhibit crucial steps of the angiogenesis, eg, the sprouting of new vessels from preexisting vessels,21,22 we hypothesized that isoprostanes may contribute to vascular rarefaction by inhibiting angiogenesis via activation of the TBXA2R.
The data presented herein provide evidence that isoprostanes inhibit vascular endothelial growth factor (VEGF)-induced endothelial cell migration, tube formation, and cardiac angiogenesis in vitro, as well as angiogenesis in vivo, via activation of the TBXA2R.
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Materials and Methods |
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Several methods, such as assessment of endothelial cell migration, the cardiac angiogenesis assay, and the chorioallantoic membrane (CAM) angiogenesis assay, have been described previously.23–26 An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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Influence of 8-Iso-PGF2
and Thromboxane A2 Mimetic U-46119 on Basal Migration of Endothelial CellsTo explore the effect of isoprostanes on basal migration of endothelial cells, we performed transwell migration assays using increasing concentrations of 8-iso-PGF2
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A, 8-iso-PGF2 exerted a biphasic effect on the basal migration of human dermal microvascular endothelial cells (HDMECs) with a moderate stimulation of migration at lower concentrations (122.1±9.9% [10–7 mol/L]; P<0.05 versus vehicle) and an inhibitory effect at higher concentrations (72.1±11.2% [10–4 mol/L]; P<0.01 versus vehicle). This biphasic response to 8-iso-PGF2 was also detected in human coronary artery endothelial cells (HCAECs) and was mimicked by the isoprostane 8-iso-PGA2 (data not shown). Moreover, the 8-iso-PGF2–induced biphasic modification of HDMEC cell migration was completely abolished in the presence of the TBXA2R antagonist SQ-29548 (Figure 1A). In contrast to 8-iso-PGF2, the thromboxane A2 mimetic U-46119 dose-dependently reduced HDMEC migration, with a maximum at the highest concentration tested (50.7±6.3% [3x10–5 mol/L]; P<0.001 versus vehicle). This effect again was reversed by concomitant incubation with SQ-29548. To elucidate the potential role of Rho kinase in isoprostane-mediated effects on HDMEC migration, experiments were repeated in presence of the Rho kinase inhibitor Y-27632 (10–5 mol/L). Interestingly, Y-27632 almost completely abolished the inhibitory effect of 8-iso-PGF2 and U-46119 on basal HDMEC migration (Figure 1C). Moreover, Y-27632 abolished the stimulatory effect of 8-iso-PGF2 on basal HDMEC migration (data not shown).
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P<0.01 vs control, **P<0.01 vs 8-iso-PGF2
Influence of 8-Iso-PGF2, 8-Iso-PGA2, and U-46119 on VEGF-Induced Migration of Endothelial Cells
Because under in vivo conditions, VEGF plays a dominant role in the process of angiogenesis, we subsequently examined the effect of isoprostanes on VEGF-induced endothelial cell migration. HDMECs were subjected to transwell migration assays and incubated with VEGF (50 ng/mL) in the presence of increasing concentrations of 8-iso-PGF2 (Figure 1B). 8-Iso-PGF2 significantly inhibited the motogenic effect of VEGF in a concentration-dependent manner. A significant inhibition was observed starting at low concentrations of 10–9 mol/L 8-iso-PGF2 (162.3±20.0% versus 185.7±14.2 [VEGF alone]; P<0.01 versus VEGF; Figure 1B), which concentration-dependently increased (125.1±9.8% versus 185.7±14.2 [VEGF alone]; P<0.001). Again, this inhibitory effect of 8-iso-PGF2 was mimicked by 8-iso-PGA2, as well as U-46119, and was also present in HCAECs (133.2±6.1% [8-iso-PGF2], 127.4±8.9% [8-iso-PGA2], 108.7±7.3% [U-46119] versus 161.3±4.5% (VEGF), P<0.001 versus VEGF, respectively; Figure 2A). Moreover, the inhibitory effect was almost completely reversed by the TBXA2R antagonist SQ-29548 (Figures 1B and 2A). SQ-29548 itself had no significant effect on basal or VEGF-induced migration of HDMECs or HCAECs (data not shown). Moreover, the inhibitory effects of 8-iso-PGF2 and U-46119 were not attributable to cytotoxic effects in HDMECs (Figure 2B). However, 8-iso-PGF2a marginally but significantly induced apoptosis of HCAECs treated with 50 ng/mL VEGF as compared to those treated with VEGF alone (15.7±1.3% versus 11.9±1.3%; P<0.05; n=8/8), an effect that was, again, partially reversed by the TBXA2R antagonist SQ-29548 (13.3±1.4%). Knockdown of TBXA2R by lentiviral short hairpin RNA selectively abolished the inhibitory effect of 8-iso-PGF2 on VEGF-induced migration of endothelial cells, whereas transduction with scrambled short hairpin RNA had no effect (Figure IA in the online data supplement). The efficacy of lentiviral knockdown was confirmed by quantitative PCR and Western blot analysis (supplemental Figures IC and ID). To investigate whether isoprostane-mediated inhibition of VEGF-induced migration was caused by isoprostane-induced production of thromboxane A2, we repeated the experiments after preincubation and in presence of the specific thromboxane A2 synthase inhibitor ozagrel (3x10–5 mol/L). However, ozagrel did not alter the inhibitory effect of isoprostanes (8-iso-PGF2 and 8-iso-PGA2) on VEGF-induced HDMEC migration (data not shown). Because activation of Rho kinase was shown to play a key role in the 8-iso-PGF2–mediated inhibition of basal EC migration, we also investigated the effect of the Rho kinase inhibitor Y-27632 on isoprostane-mediated inhibition of VEGF-induced HDMEC migration (Figure 1D). Again Y-27632 strongly attenuated the inhibitory effect of 8-iso-PGF2 and U-46119 on VEGF-induced HDMEC migration, thereby indicating that VEGF-induced migration depends on a concerted activation of Rho kinase. In contrast, Y-27632 itself had no effect on basal or VEGF-stimulated endothelial cell migration (supplemental Figure IIIB).
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Influence of 8-Iso-PGF2, 8-Iso-PGA2, and U-46119 on VEGF-Induced Tube Formation of HCAECs In Vitro
Also, we investigated the influence of 8-iso-PGF2, 8-iso-PGA2, 8-iso-PGE2, and U-46119 on VEGF-induced tube formation of HCAECs, a further essential step in the angiogenic process. In this Matrigel-based assay, endothelial tube formation was induced by treatment with VEGF (20 ng/mL; Figure 3). Addition of 8-iso-PGF2, 8-iso-PGA2, 8-iso-PGE2, and U-46119 (3x10–5 mol/L, respectively) reduced VEGF-induced tube formation of HCAECs (81±13% [8-iso-PGF2], 74±10% [8-iso-PGA2], 84±11% [8-iso-PGE2], 54±13% [U-46119] versus 118±16% [VEGF]; P<0.001 versus VEGF, respectively; Figure 3C). This inhibitory effect was again resolved by the TBXA2R antagonist SQ-29548, short hairpin RNA–mediated knockdown of TBXA2R, and the Rho kinase inhibitor Y-27632 (supplemental Figures IA and IIIB). Moreover, simultaneous addition of 8-iso-PGF2, 8-iso-PGA2, and 8-iso-PGE2 (3x10–5 mol/L, respectively) resulted in a significantly stronger inhibition of endothelial cell tube formation compared with the inhibition induced by any of the isoprostanes alone (54±9%; P<0.01 versus 8-iso-PGF2, 8-iso-PGA2, 8-iso-PGE2 alone) indicating a synergistic inhibitory effect of isoprostanes on tube formation of endothelial cells in vitro.
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Decomposition of the Isoprostane 8-Iso-PGA2 Into Two Biologically Active Compounds, X and Y, In Vitro
In contrast to the isoprostane 8-iso-PGF2, which has been shown to be chemically stable, the stability of further isoprostanes, such as 8-iso-PGA2, remained elusive. Hence, we investigated the stability of 8-iso-PGA2 under "physiological" conditions (pH 7.4, 37°C) in vitro (Figure 4A through 4C). Most interestingly, we observed a decomposition of 8-iso-PGA2 within 24 hours into an unknown compound, termed X, which, again, completely transformed within 24 hours into compound Y (Figure 4A through 4C). Liquid chromatography–tandem mass spectrometric analysis of compound X revealed a molecular ion [M-H]– of m/z 333 and the presence of the fragmentation series m/z 315, m/z 271, and m/z 217 (Figure 4E), corresponding to loss of water, subsequent decarboxylation, and cleavage at the Z-double bound, respectively. The fragmentation ions m/z 271 and m/z 217, together with the molecular ion [M-H]– m/z 315, were also found for compound Y and the cyclopentenone isomer 15-deoxy-PGJ2 (Figure 5F and 5G). From the GC-MS analysis of the PFB-MO-(TMS) derivatives of 8-iso-PGE2, compounds X and Y, the most abundant ions were m/z 524, m/z 434, and m/z 344, respectively ([M-PFB]–). A potential chemical fate of compounds investigated is outlined in Figure 4H. To test biological activity of both compounds, the effect on VEGF-induced tube formation of HCAECs was investigated. Surprisingly, both compounds exerted potent inhibitory effects in this setting (77±4% [X], 68±4% [Y] versus 119±5% [VEGF]; P<0.001 versus VEGF, respectively), which, again, was partly reversed by the TBXA2R antagonist SQ-29548 (Figure 4D). These findings strongly suggest that specific instable isoprostanes may nonenzymatically form biologically active derivatives in vivo, which may exert synergistic effects together with further endogenous isoprostanes via the TBXA2R.
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12,14-prostaglandin J2 (15-deoxy-PGJ2). H, Proposed chemical fate of 8-iso-PGA2 through a sequence of isomerization and dehydration reactions.
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Effect of 8-Iso-PGF2 and U-46119 on Cardiac Angiogenesis In Vitro
To further elucidate the role of 8-iso-PGF2 and U-46119 in the process of angiogenesis, we chose an experimental that which is more closely related to the in vivo situation (Figure 6). Interestingly, both 8-iso-PGF2 and U-46119 (at 3x10–5 mol/L, respectively) attenuated hypoxia-driven VEGF and platelet-derived growth factor (PDGF)-induced cardiac angiogenesis in vitro (Figure 6C and 6D). Again, this inhibitory effect of both compounds was almost completely reversed by the TBXA2R antagonist SQ-29548 (Figure 6E and 6F). In contrast, low concentrations of 8-iso-PGF2 applied in the absence of VEGF and PDGF did not affect hypoxia-driven cardiac angiogenesis in our in vitro system (data not shown).
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Effect of 8-Iso-PGF2 and U-46119 on VEGF-Induced Angiogenesis in the CAM Assay In Vivo
The CAM assay was chosen to elucidate the effect of 8-iso-PGF2 on VEGF-induced angiogenesis in vivo. In comparison to the control, in which only PBS was added, the application of VEGF induced a strong vascularization as it is recognizable by the intense vascular branching under the mesh (Figure 5). In contrast, the simultaneous application of VEGF and 8-iso-PGF2 or the thromboxane A2 mimetic U-46619 blocked the VEGF-induced neovascularization. The combined application of VEGF and the specific TBXA2R antagonists SQ-29548 did not alter the angiogenic effect of VEGF. The inhibitory effect of 8-iso-PGF2 was partially reversed when VEGF and 8-iso-PGF2 were applied simultaneously with SQ-29548. Similar effects were observed in the case of combined application of VEGF in presence of U-46619 and SQ-29548.
Effect of 8-Iso-PGF2 on Basal and VEGF-Induced Akt and Extracellular Signal-Regulated Kinase and Focal Adhesion Kinase Signaling in HDMECs
As Akt has been shown to affect endothelial cell motility, we investigated the effect of 8-iso-PGF2 on basal as well as VEGF-induced Akt (Ser-473) phosphorylation in HDMECs. Interestingly, lower promigratory concentrations of 8-iso-PGF2 (10–7 mol/L) induced basal phosphorylation of Akt (Figure 7A). Again, TBXA2R antagonist SQ-29548, which abolished the promigratory effect of low-concentrated 8-iso-PGF2, also reversed the 8-iso-PGF2–induced Akt phosphorylation (Figure 7A). Moreover, HDMECs treated with high (antimigratory) concentrations of 8-iso-PGF2 (3x10–5 mol/L) exhibited levels of phosphorylated Akt comparable to those of unstimulated control cells (Figure 7A). In contrast, extracellular signal-regulated kinase (ERK)-1/-2 (Thr202/Tyr204) phosphorylation, dose-dependently increased in HDMECs treated with 8-iso-PGF2, an effect that was sensitive to the TBXA2R antagonist SQ-29548 (Figure 7A). However, 8-iso-PGF2 did not significantly affect VEGF-induced Akt, ERK, or focal adhesion kinase (FAK) (pY397) phosphorylation after 15 minutes of costimulation (Figure 7B; p-FAK data not shown).
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Effect of 8-Iso-PGF2 on Endothelial Cell Apoptosis
To investigate the effect of 8-iso-PGF2a on endothelial cell apoptosis, we flow-cytometrically assessed annexin V–fluorescein isothiocyanate (FITC) binding to stimulated HCAECs. After 12 hours slightly in this setting, 8-iso-PGF2 but significantly induced apoptosis of HCAECs treated with VEGF (50 ng/mL; Figure 2C and 2D) as compared to those treated with VEGF alone (15.7±1.3% versus 11.9±1.3%; P<0.05; n=8/8), an effect that was, again, partially reversed by the TBXA2R antagonist SQ-29548 (13.3±1.4%).
Effect of 8-Iso-PGF2 and U-46119 on Generation of F-Actin Stress Fibers in HDMECs
Stress fiber formation, which is critical for endothelial cell movement, has been shown to be regulated by a concerted activation of Rho kinase.27 Thus, we investigated the influence of 8-iso-PGF2 and U-46119 on generation of F-actin stress fibers in HDMECs. HDMECs treated with VEGF (50 ng/mL, 4 hour; Figure 8B) showed increased formation of stress fibers as compared with untreated HDMECs (Figure 8A). 8-Iso-PGF2 and U-46119 (3x10–5 mol/L, respectively) interfered with the VEGF-induced effects and induced a nondirectional stress fiber formation and focal adhesion pattern (Figure 8C and 8E). This effect could be reversed by concomitant stimulation with SQ-29548 (Figure 8D and 8F).
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Discussion |
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The main novel findings of the present study are that isoprostanes concentration-dependently inhibit VEGF-induced migration and tube formation of endothelial cells as well as VEGF-induced cardiac angiogenesis in vitro and angiogenesis in vivo via activation of the TBXA2R. In this regard, different isoprostanes exerted a synergistic inhibitory effect on endothelial cell tube formation. Moreover, spontaneously formed derivatives of 8-iso-PGA2, termed compound X and Y, were biologically active and inhibited in vitro tube formation of HCAECs. Furthermore, the inhibitory effect of isoprostanes on VEGF-induced angiogenesis was sensitive to pharmacological inhibition of Rho kinases, thereby indicating involvement of Rho kinases in the antiangiogenic effect of isoprostanes.
Angiogenesis, the formation of new blood capillaries, is of crucial importance for the pathophysiology of multiple diseases, including myocardial ischemia in patients with CHD.19 The growth of neovessels is a tightly regulated process that is controlled by the concerted release of pro- and antiangiogenic factors.19 VEGF has emerged as the key regulator of angiogenesis and promotes most of the critical steps of this process, such as endothelial cell movement, proliferation, and capillary tube formation.28 Most interestingly, VEGF is elevated in the serum of patients with myocardial ischemia and may stimulate neovascularization of the ischemic myocardium and promote the development of collateral vessels to restore or maintain myocardial blood flow.17,18 Hence, antiangiogenic stimuli counteracting VEGF-induced revascularization processes may negatively affect these essential hypoxia-driven adaptive changes. In the present study, we clearly demonstrate that isoprostanes, endogenously formed end products of free radical–induced lipid peroxidation, inhibit VEGF-induced migration and tube formation of ECs, as well as cardiac angiogenesis in vitro and angiogenesis in vivo. In this regard, findings of our group (and others) that isoprostanes accumulate in patients with CHD and in patients at high cardiovascular risk, eg, hypertensive individuals, clearly underline the clinical importance of our experimental findings. Moreover, recently published data demonstrate that isoketals, which form nonenzymatically from the same precursors (H2-isoprostane regioisomers) in vivo as the 8-isoprostanes investigated in our study, considerably accumulate in hypoxic myocardium.12 These findings strongly suggest that elevated systemic concentrations of isoprostanes observed in patients with CHD (stable CHD patients display a 2- to 3-fold increase in systemic isoprostane concentrations, which are in the concentration range of 30 to 40 ng/L in the plasma of healthy humans29) may not adequately reflect enhanced local generation of isoprostanes in hypoxic tissues. Moreover, isoprostane metabolites and so far unknown but biologically active decomposition products of specific isoprostanes (eg, cyclopentenone isoprostane derivatives X and Y), which are not captured in the present analytic routines for isoprostane detection, may accumulate in patients at high cardiovascular risk and synergistically exert antiangiogenic effects (as shown in this study for the isoprostanes 8-iso-PGF2, 8-iso-PGA2, and 8-iso-PGE2). Hence, isoprostanes may contribute to development of capillary rarefaction, which has been observed in pathophysiological states of increased oxidative stress, such as CHD or systemic hypertension.19,20
In our study, 8-iso-PGF2, 8-iso-PGA2, and 8-iso-PGE2 inhibited the VEGF-induced angiogenic response via activation of the TBXA2R, because the TBXA2R antagonists SQ-29548, BM 567, and ICI 192,605 reversed isoprostane-induced inhibition of angiogenesis (online data supplement). Besides pharmacological antagonism of the TBXA2R, we were able to demonstrate that lentiviral knockdown of TBXA2R also resolved the inhibitory effect of 8-iso-PGF2 (online data supplement). Our findings are in line with other studies that demonstrate antiangiogenic actions of this receptor,21,22,30 although 1 group described proangiogenic effects of TBXA2R activation.31 The present study identifies for the first time isoprostanes as important endogenous compounds, which are capable of exerting antiangiogenic effects via activation of the TBXA2R. Moreover, isoprostanes slightly but significantly induced apoptosis in endothelial cells via activation of the TBXA2R, an effect that may partially contribute to the antiangiogenic properties of the compounds. Previously, a proapoptotic effect of isoprostanes on neuromicrovascular endothelial cells has been described.32 In addition to their antiangiogenic actions, isoprostanes may, by this mechanism, contribute to endothelial dysfunction in disease states of increased oxidative stress.33
In contrast to the findings of previous publications,21,22 our results do not point to an inhibitory effect of the thromboxane A2 mimetic U-46119 or 8-iso-PGF2 on VEGF-induced protein kinase B/Akt (Ser-473) or FAK (pY397) phosphorylation in endothelial cells. The reason for this discrepancy may be ascribed to different cell types studied (HDMECs versus HUVECs) but remain uncertain. Moreover, U-46119 or 8-iso-PGF2 did not significantly affect VEGF-induced ERK-1/-2 phosphorylation after 15 minutes of costimulation in HDMECs. However, the inhibitory effect of 8-iso-PGF2 and U-46119 was almost completely abolished by coincubation with the Rho kinase inhibitor Y-27632, thereby indicating that a pronounced and persistent TBXA2R-mediated Rho kinase activation may be responsible for the isoprostane-induced inhibition of basal and VEGF-stimulated endothelial cell migration and tube formation. This perception is supported by detection of an increased and more persistent RhoA activation induced by concomitant stimulation with 8-iso-PGF2a in endothelial cells as compared to the observed transient RhoA activation induced by VEGF alone (online data supplement). Moreover, very recent data from Wikström et al support the notion that activation of the TBXA2R mediates a persistent activation of RhoA, which, in turn, inhibits depolymerization of F-actin.34 In this regard, RhoA and downstream Rho kinases are known to play a crucial role in endothelial cell motility by regulating the formation of F-actin stress fibers as well as focal adhesion turnover.35 Hence, RhoA-mediated inhibition of F-actin depolymerization could disturb the concerted dynamics of F-actin reorganization, which is necessary for cell movement. Indeed, histological analysis of 8-iso-PGF2– and U-46119–treated HDMECs revealed that these substances disturbed the directional and concerted VEGF-induced stress fiber formation and focal adhesion formation. Moreover, new findings suggest that persistent Rho kinase activity may inhibit endothelial cell motility of endothelial cells by increasing cell adhesion to the substratum or slowing down turnover of focal adhesions.27 In addition, Rho kinase activation may counteract angiogenesis in ischemic tissues by mediating downregulation of endothelial NO synthase.36
8-Iso-PGF2 and 8-iso-PGA2 exerted a biphasic response on basal migration of HDMECs and HCAECs, respectively, with lower concentrations moderately inducing basal EC migration. In line with these observations, lower promigratory concentrations of 8-iso-PGF2 induced basal Akt phosphorylation in HDMECs, whereas higher antimigratory concentrations did not. In contrast, ERK-1/-2 phosphorylation was shown to be concentration-dependently induced by 8-iso-PGF2. These effects were mediated via TBXA2R activation because SQ-29548 abrogated 8-iso-PGF2–induced promigratory effects and changes in HDMEC signaling transduction. Moreover, Rho kinase inhibitor Y-27632 abolished the stimulatory effect of 8-iso-PGF2 on basal HDMEC migration. These results suggest that lower concentrations of 8-iso-PGF2 are able to induce concerted activation of Akt and Rho kinase via activation of the TBXA2R and by this means moderately stimulate basal endothelial cell motility in the absence of VEGF (a scenario unlikely to occur in vivo because VEGF and other growth factors are constantly secreted under physiological conditions), whereas 8-iso-PGF2 (and other isoprostanes tested) concentration-dependently inhibit VEGF-induced EC migration. Moreover, promigratory concentrations of 8-iso-PGF2a did not induce basal cardiac angiogenesis in vitro. Hence, the physiological relevance of isoprostane-induced basal endothelial cell migration remains unclear.
In conclusion, our study demonstrates for the first time that isoprostanes exert an inhibitory effect on VEGF-induced migration and capillary tube formation of endothelial cells, as well as on cardiac angiogenesis in vitro and angiogenesis in vivo via activation of the TBXA2R. Taken together, our findings uncover mechanisms by which isoprostanes may exert antiangiogenic actions especially in disease states of increased oxidative stress and elevated isoprostane levels.
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Acknowledgments |
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Sources of Funding
This work was supported by Werner Otto foundation grant Be 6/70 (Hamburg, Germany).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received November 21, 2007; resubmission received July 28, 2008; revised resubmission received September 4, 2008; accepted September 8, 2008.
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References |
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1. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
2. Richter C, Gogvadze V, Laffranchi R, Schlapbach R, Schweizer M, Suter M, Walter P, Yaffee M. Oxidants in mitochondria: from physiology to diseases. Biochim Biophys Acta. 1995; 1271: 67–74.[Medline] [Order article via Infotrieve]
3. Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci U S A. 1990; 87: 9383–9387.
4. Montuschi P, Barnes P, Roberts LJ II. Insights into oxidative stress: the isoprostanes. Curr Med Chem. 2007; 14: 703–717.[CrossRef][Medline] [Order article via Infotrieve]
5. Schwedhelm E, Bartling A, Lenzen H, Tsikas D, Maas R, Brummer J, Gutzki FM, Berger J, Frolich JC, Boger RH. Urinary 8-iso-prostaglandin F2 as a risk marker in patients with coronary heart disease: a matched case-control study. Circulation. 2004; 109: 843–848.
6. Morrow JD, Frei B, Longmire AW, Gaziano JM, Lynch SM, Shyr Y, Strauss WE, Oates JA, Roberts LJ II. Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers. Smoking as a cause of oxidative damage. N Engl J Med. 1995; 332: 1198–1203.
7. Davi G, Alessandrini P, Mezzetti A, Minotti G, Bucciarelli T, Costantini F, Cipollone F, Bon GB, Ciabattoni G, Patrono C. In vivo formation of 8-Epi-prostaglandin F2 is increased in hypercholesterolemia. Arterioscler Thromb Vasc Biol. 1997; 17: 3230–3235.
8. Reilly MP, Delanty N, Roy L, Rokach J, Callaghan PO, Crean P, Lawson JA, FitzGerald GA. Increased formation of the isoprostanes IPF2-I and 8-epi-prostaglandin F2 in acute coronary angioplasty: evidence for oxidant stress during coronary reperfusion in humans. Circulation. 1997; 96: 3314–3320.
9. Laffer CL, Bolterman RJ, Romero JC, Elijovich F. Effect of salt on isoprostanes in salt-sensitive essential hypertension. Hypertension. 2006; 47: 434–440.
10. Il'yasova D, Morrow JD, Wagenknecht LE. Urinary F2-isoprostanes are not associated with increased risk of type 2 diabetes. Obes Res. 2005; 13: 1638–1644.[Medline] [Order article via Infotrieve]
11. Davi G, Ciabattoni G, Consoli A, Mezzetti A, Falco A, Santarone S, Pennese E, Vitacolonna E, Bucciarelli T, Costantini F, Capani F, Patrono C. In vivo formation of 8-iso-prostaglandin f2 and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation. Circulation. 1999; 99: 224–229.
12. Fukuda K, Davies SS, Nakajima T, Ong BH, Kupershmidt S, Fessel J, Amarnath V, Anderson ME, Boyden PA, Viswanathan PC, Roberts LJ II, Balser JR. Oxidative mediated lipid peroxidation recapitulates proarrhythmic effects on cardiac sodium channels. Circ Res. 2005; 97: 1262–1269.
13. Schwedhelm E, Benndorf RA, Boger RH, Tsikas D. Mass spectrometric analysis of F2-isoprostanes: markers and mediators in human disease. Curr Pharm Anal. 2007; 1: 39–51.
14. Hou X, Roberts LJ II, Gobeil F Jr, Taber D, Kanai K, Abran D, Brault S, Checchin D, Sennlaub F, Lachapelle P, Varma D, Chemtob S. Isomer-specific contractile effects of a series of synthetic F2-isoprostanes on retinal and cerebral microvasculature. Free Radic Biol Med. 2004; 36: 163–172.[CrossRef][Medline] [Order article via Infotrieve]
15. Tang M, Cyrus T, Yao Y, Vocun L, Pratico D. Involvement of thromboxane receptor in the proatherogenic effect of isoprostane F2-III: evidence from apolipoprotein E- and LDL receptor-deficient mice. Circulation. 2005; 112: 2867–2874.
16. Folco G, Granstrom E, Kindahl H. Albumin stabilizes thromboxane A2. FEBS Lett. 1977; 82: 321–324.[CrossRef][Medline] [Order article via Infotrieve]
17. Schaper W, Ito WD. Molecular mechanisms of coronary collateral vessel growth. Circ Res. 1996; 79: 911–919.
18. Lee SH, Wolf PL, Escudero R, Deutsch R, Jamieson SW, Thistlethwaite PA. Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N Engl J Med. 2000; 342: 626–633.
19. Carmeliet P. Angiogenesis in life, disease and medicine. Nature. 2005; 438: 932–936.[CrossRef][Medline] [Order article via Infotrieve]
20. Ciuffetti G, Schillaci G, Innocente S, Lombardini R, Pasqualini L, Notaristefano S, Mannarino E. Capillary rarefaction and abnormal cardiovascular reactivity in hypertension. J Hypertens. 2003; 21: 2297–2303.[CrossRef][Medline] [Order article via Infotrieve]
21. Gao Y, Yokota R, Tang S, Ashton AW, Ware JA. Reversal of angiogenesis in vitro, induction of apoptosis, and inhibition of AKT phosphorylation in endothelial cells by thromboxane A(2). Circ Res. 2000; 87: 739–745.
22. Ashton AW, Ware JA. Thromboxane A2 receptor signaling inhibits vascular endothelial growth factor-induced endothelial cell differentiation and migration. Circ Res. 2004; 95: 372–379.
23. Benndorf R, Boger RH, Ergun S, Steenpass A, Wieland T. Angiotensin II type 2 receptor inhibits vascular endothelial growth factor-induced migration and in vitro tube formation of human endothelial cells. Circ Res. 2003; 93: 438–447.
24. Kiefer FN, Munk VC, Humar R, Dieterle T, Landmann L, Battegay EJ. A versatile in vitro assay for investigating angiogenesis of the heart. Exp Cell Res. 2004; 300: 272–282.[CrossRef][Medline] [Order article via Infotrieve]
25. Iruela-Arispe ML, Lane TF, Redmond D, Reilly M, Bolender RP, Kavanagh TJ, Sage EH. Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo. Mol Biol Cell. 1995; 6: 327–343.[Abstract]
26. Ergün S, Kilik N, Ziegeler G, Hansen A, Nollau P, Götze J, Wurmbach JH, Horst A, Weil J, Fernando M, Wagener C. CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor. Mol Cell. 2000; 5: 311–320.[CrossRef][Medline] [Order article via Infotrieve]
27. Wojciak-Stothard B, Torondel B, Tsang LY, Fleming I, Fisslthaler B, Leiper JM, Vallance P. The ADMA/DDAH pathway is a critical regulator of endothelial cell motility. J Cell Sci. 2007; 120: 929–942.
28. Zachary I. Signal transduction in angiogenesis. EXS. 2005; 94: 267–300.[Medline] [Order article via Infotrieve]
29. Milne GL, Sanchez SC, Musiek ES, Morrow JD. Quantification of F2-isoprostanes as a biomarker of oxidative stress. Nat Protoc. 2007; 2: 221–226.[CrossRef][Medline] [Order article via Infotrieve]
30. Pal S, Wu J, Murray JK, Gellman SH, Wozniak MA, Keely PJ, Boyer ME, Gomez TM, Hasso SM, Fallon JF, Bresnick EH. An antiangiogenic neurokinin-B/thromboxane A2 regulatory axis. J Cell Biol. 2006; 174: 1047–1058.
31. Nie D, Lamberti M, Zacharek A, Li L, Szekeres K, Tang K, Chen Y, Honn KV. Thromboxane A(2) regulation of endothelial cell migration, angiogenesis, and tumor metastasis. Biochem Biophys Res Commun. 2000; 267: 245–251.[CrossRef][Medline] [Order article via Infotrieve]
32. Brault S, Martinez-Bermudez AK, Marrache AM, Gobeil F Jr, Hou X, Beauchamp M, Quiniou C, Almazan G, Lachance C, Roberts J II, Varma DR, Chemtob S. Selective neuromicrovascular endothelial cell death by 8-Iso-prostaglandin F2alpha: possible role in ischemic brain injury. Stroke. 2003; 34: 776–782.
33. Belhassen L, Pelle G, Dubois-Rande JL, Adnot S. Improved endothelial function by the thromboxane A2 receptor antagonist S 18886 in patients with coronary artery disease treated with aspirin. J Am Coll Cardiol. 2003; 41: 1198–1204.
34. Wikström K, Kavanagh DJ, Reid HM, Kinsella BT. Differential regulation of RhoA-mediated signaling by the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor: independent modulation of TPalpha signaling by prostacyclin and nitric oxide. Cell Signal. 2008; 20: 1497–1512.[CrossRef][Medline] [Order article via Infotrieve]
35. Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res. 2007; 100: 782–794.
36. Takemoto M, Sun J, Hiroki J, Shimokawa H, Liao JK. Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation. 2002; 106: 57–62.
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