Angiotensin II Inhibits Endothelial Cell Motility Through an AT1-Dependent Oxidant-Sensitive Decrement of Nitric Oxide Availability
Objective— The migratory capability of vascular endothelial cells plays a pivotal role in the maintenance of vessel wall integrity and is stimulated by nitric oxide (NO). Angiotensin II increases NAD(P)H oxidase activity in endothelial cells, thereby promoting reactive oxygen species (ROS) generation. Because ROS can both reduce NO synthase activity and increase NO breakdown, thus impairing NO availability in endothelial cells, we evaluated the effect of angiotensin II on human vascular endothelial cell (HUVEC) motility.
Methods and Results— Angiotensin II dose- and time-dependently reduced HUVEC migration. Besides inhibiting HUVEC motility, angiotensin II altered intracellular glutathione redox status. The generation of ROS by cultured HUVECs was significantly increased by angiotensin II. Furthermore, angiotensin II reduced NO metabolite concentrations in culture media. The angiotensin II type 1 receptor antagonist candesartan cilexetil attenuated the inhibitory action exerted by angiotensin II on HUVEC motility, reversed the angiotensin II-induced increase in intracellular oxidative stress, and restored NO availability. Similar effects were exerted by the flavonoid inhibitor diphenylene iodinium and the antioxidant agent N-acetyl-l-cysteine.
Conclusions— All together, our data demonstrate that angiotensin II inhibits HUVEC motility by reducing NO availability. Such reduction is due to an angiotensin II type 1 receptor-dependent increment in intracellular ROS generation.
The rapid migration of proliferating endothelial cells to reconstitute a continuous endothelial monolayer is fundamental for the restoration of vessel wall integrity.1–3⇓⇓ Different pathologic conditions could be associated with abnormalities in the migratory capability of vascular endothelial cells, including human atherosclerosis and restenosis occurring after percutaneous transluminal coronary angioplasty either with or without arterial stenting.4
In this context, several studies have demonstrated that the angiotensin system modulates the response of vascular endothelial and smooth muscle cells to injury.5 With respect to endothelial cell motility, previous reports have shown that angiotensin II can influence endothelial cell migratory ability.6,7⇓ Furthermore, an autocrine pathway leading to angiotensin II formation has been demonstrated in endothelial cells, where it modulates their migratory response to various stimuli.7
Mechanisms underlying the angiotensin II action on vascular endothelial cell motility are not fully understood. Angiotensin II strongly affects intracellular oxidative stress.5,8⇓ Indeed, the peptide can activate membrane-bound NAD(P)H oxidase, with a consequent increment in the production of superoxide anion (·O2−).9 In vascular endothelial cells, ·O2− can be converted by superoxide dismutase to hydrogen peroxide, which, if unscavenged by catalases or peroxidases, can deeply influence several endothelial functions, including cell proliferation and migration.5,9,10⇓⇓ In addition, ·O2− quenches nitric oxide (NO) to give peroxynitrite,11 one of the most potent endocellular oxidants,8,11⇓ and thereby reduces NO availability. Peroxynitrite also oxidizes tetrahydrobiopterin, a critical cofactor for NO synthase, thus additionally decreasing NO production.12
The oxidant properties of angiotensin II have been suggested to influence several endothelial functions.5 In particular, an angiotensin II–mediated increment in intracellular oxidative stress participates in the upregulation of redox-sensitive genes codifying for the synthesis of chemoattractant proteins as well as for the expression of adhesion molecules on the endothelial cell membrane.5,10⇓ Such upregulation could promote atherogenesis by favoring leukocyte/monocyte adhesion to the luminal endothelial cell surface and the consequent transendothelial migration of these circulating cells into the vessel wall. In addition, the pro-oxidant properties of angiotensin II could lead to the oxidation of arachidonic acid and thereby induce the release of the potent vasoconstrictor substance 8-iso-prostaglandin F2α.5,10⇓
In the present study, we investigated the impact of angiotensin II–mediated changes in intracellular oxidative stress on human vascular endothelial cell motility by using a modified Boyden chamber system. We demonstrated that angiotensin II reduced vascular endothelial cell motility through angiotensin II type 1 receptor (AT1) stimulation. We additionally investigated the underlying signaling pathway and showed that the inhibitory action exerted by angiotensin II on vascular endothelial cell motility was mediated by a reduction in NO availability. In turn, this latter reduction was attributable to an AT1-dependent increase in intracellular oxidative stress.
Human vascular endothelial cells (HUVECs) were harvested from fresh human umbilical cord veins and cultured until the third passage, as previously described.13
Purity of the endothelial cell monolayer was confirmed by the cobblestone morphological pattern and by cell staining with a monoclonal antibody specific for von Willebrand factor.14
Newly confluent cells were lifted with trypsinization; the trypsin was inhibited with 20% FCS, and cells were washed in culture medium. After centrifugation (1100 rpm over a period of 10 minutes at +20°C), the supernatant was removed and HUVECs were resuspended in culture medium (3 mL). Finally, HUVECs were used for measurements of motility, NO production, glutathione (GSH) redox status, and oxidative stress. All of the above parameters were evaluated after 18 hours incubation with angiotensin II (10−7 mol/L). Angiotensin II was added to culture media alone or with either the AT1 receptor antagonist candesartan cilexetil (10−7 mol/L), the AT2 receptor antagonist PD 123319 (10−7 mol/L), the phospholipase D (PLD) inhibitor dihydro-d-erythro-sphingosine (SPH, 10−7 mol/L), the flavoprotein inhibitor diphenylene iodinium (DPI, 10−7 mol/L), or the antioxidant N-acetyl-cysteine (NAC, 10−7 mol/L). In additional experiments, the effects of angiotensin II on HUVEC migratory ability, NO production, GSH redox status, and oxidative stress were also evaluated after either 3 or 18 hours of incubation with angiotensin II, in both cases by using different angiotensin II concentrations (10−9, 10−7, or 10−6 mol/L).
Angiotensin II was purchased from Clinalfa. Candesartan cilexetil was kindly donated by Takeda Spa, Italy. All other reagents were purchased from Sigma Chemical Co.
Endothelial Cell Migration Assay
In vitro endothelial cell migration assay was performed according to the modified Boyden chamber system. Briefly, cell culture inserts (diameter, 10 mm; 8.0-μm pore size gelatinized polycarbonate membrane, NUNC) were placed into a well of a 24-well multidish with the membrane toward the well bottom, ensuring no air was trapped. Endothelial cell suspension was added to the interior of each insert while grown media was added to the well containing insert. After 3- and 18-hour incubation at +37°C, cells adherent to the polycarbonate membrane were fixed with 95% ethanol for 8 minutes and stained by Giemsa. Then each insert was disassembled, and the top of the membrane was scraped to remove nonmigrated HUVECs. The number of migrated cells on the lower face of the filter was counted in 3 random microscopic fields under a ×20 magnification. All assays were done in triplicate and repeated each time at least twice. Cell viability was>95% (by Tripan blue exclusion) for each condition.
Measurement of NO Production
NO production by HUVECs was assessed by evaluating the concentration of NO metabolites (NOx), ie, nitrite plus nitrate, in culture media, as previously described.13 Briefly, NOx concentrations were evaluated by colorimetric detection of nitrite after conversion of all sample nitrate to nitrite (Assay Design Inc).
Measurements of Intracellular Glutathione Redox Status and Oxidative Stress
Intracellular GSH concentration was measured according to the method previously described by our group.15,16⇓ In brief, 2×106 HUVECs were diluted in 1 mL isotonic saline plus HCl (10 mmol/L) and then lysed in acetone, thawed 4 times, and centrifuged for 15 minutes at +4°C. Supernatants were deproteinized with 10% 5-sulfosalicylic acid and used for total GSH determination, ie, GSH plus GSH disulphide (GSSG), by the enzymatic method described by Anderson.17 For GSSG determination, 0.1 mL deproteinized supernatants were treated with 2 μL 2-vinylpyridine, neutralized with triethanolamine at a final pH of 6.5, and assayed after 1 hour of incubation.17
Angiotensin II–induced oxidative stress was measured according to the method by Wu and Juurlink.18 In brief, cultured HUVECs were loaded with the permeable agent 5-(6)-carboxy-2′-7′-dichlorodihydrofluorescein (DCHF) ester for 60 minutes. As is known, in the presence of intracellular esterases, permeable DCHF ester is converted to its impermeable counterpart. This latter is oxidized to the fluorescent dichlofluorescein (DCF) by strong oxidants such as hydroxyl radicals.18,19⇓ Therefore, oxidative stress in HUVECs can be quantified by monitoring intracellular DCF content by a fluorometer with excitation at 495 nm and emission at 525 and expressed as percent of control.18
HUVEC Proliferation Assay
To avoid bias attributable to angiotensin II–induced HUVEC proliferation, this latter was measured after HUVECs were incubated for 18 hours with increasing angiotensin II concentrations (10−9, 10−7, or 10−6 mol/L). For this purpose, HUVECs were cultured on 96-well flat-bottom microtiter plates and proliferation was assessed by using the MTS colorimetric assay (Cell Titer 96 Aqueous Non Radioactive Cell Proliferation Assay from Promega),20 according to manufacturer’s instructions.
Changes in migration were analyzed by ANOVA followed by Bonferroni’s test. Multiple comparisons were analyzed by ANOVA followed by post-hoc analysis to adjust the significance level. Statistical significance was considered as P<0.05. Data are given as the mean±SD of experiments conducted on 2 different occasions, each time in triplicate.
Angiotensin II Inhibited HUVEC Migration by Stimulating the AT1 Receptor
HUVEC migration was inhibited by angiotensin II in dose- and time-dependent manners (Figure 1A). The AT1 receptor antagonist candesartan cilexetil attenuated the effects of angiotensin II on HUVEC motility (Figure 1B). Candesartan cilexetil also reduced spontaneous endothelial cell migratory ability. In contrast, the angiotensin II type 2 (AT2) receptor inhibitor PD 123319 was unable to modify both spontaneous HUVEC motility and the angiotensin II actions on HUVEC migratory ability (Figure 1B).
NAD(P)H Oxidase–Related Induction of Intracellular Oxidative Stress Was Involved in the Inhibitory Actions Exerted by Angiotensin II on HUVEC Motility
Compared with untreated HUVECs, angiotensin II–stimulated ones showed significant changes in redox status. In particular, angiotensin II reduced GSH (Figure 2A) and increased GSSG (Figure 2B) contents, with a consequent increment of GSSG/GSH ratio (Figure 2C). Furthermore, angiotensin II increased oxidative stress as evaluated by intracellular DCF content (Figure 2D). The AT1 receptor inhibitor candesartan cilexetil completely prevented angiotensin II–related changes in intracellular GSH redox status and DCF content (Figures 3A through 3D). By contrast, the AT2 receptor inhibitor PD 123319 was completely ineffective in counteracting angiotensin II–related changes in oxidant/antioxidant balance (Figures 3A through 3D). Preincubation of HUVECs with the PLD inhibitor SPH partially reversed the effects of angiotensin II on intracellular oxidative status (Figures 3A through 3D). The flavoprotein inhibitor DPI almost completely abolished the effects of angiotensin II on GSH redox status and DCF content (Figures 3A through 3D). These data suggest that angiotensin II–induced changes in intracellular oxidant/antioxidant balance were related to an AT1 receptor–dependent activation of NAD(P)H or similar flavoprotein-dependent oxidase, which was only partially mediated by the PLD pathway.
When HUVECs were preincubated with DPI, the inhibitory action of angiotensin II on endothelial cell motility was significantly attenuated. The PLD inhibitor SPH only partially blocked the antimigratory effect of angiotensin II (Figure 1B). These data suggest that angiotensin II–related changes in intracellular oxidative stress were involved in the angiotensin II–induced changes in HUVEC motility. In keeping with this, the antioxidant NAC attenuated the effects of angiotensin II on endothelial cell motility (Figure 1B) and completely prevented angiotensin II–induced changes in intracellular oxidant/antioxidant balance (Figures 3A through 3D).
DPI and NAC had no additive inhibitory actions on the angiotensin II effects on HUVEC motility (data not shown). Similarly, addition of either DPI or NAC to candesartan cilexetil did not additionally potentiate the inhibitory effects of AT1 receptor blockade (data not shown). Finally, SPH did not additionally increase HUVEC motility after incubation with either candesartan cilexetil, DPI, or NAC (data not shown).
The PLD inhibitor SPH and the flavoprotein inhibitor DPI as well as the antioxidant NAC also affected spontaneous HUVEC motility (Figure 1B), intracellular oxidant/antioxidant balance (Figures 3A through 3D), and NO availability (Figure 4B).
As is known, the flavoprotein inhibitor used in our experiments to block NAD(P)H oxidase, DPI, also inhibits xanthine oxidase activity. To avoid this bias, we tested the effects of the specific xanthine oxidase inhibitor allopurinol on HUVEC motility. Allopurinol did not significantly affect both spontaneous and angiotensin II–stimulated endothelial cell motility. These findings excluded the participation of xanthine oxidase activity in the antimigratory action exerted by angiotensin II.
Impaired NO Availability Was Responsible for the Inhibitory Effect of Angiotensin II on HUVEC Motility
NO plays a fundamental role in regulating endothelial cell migratory capability.21 Angiotensin II–related changes in oxidative stress could affect NO availability by increasing NO breakdown22 and reducing NO synthase activity.12 In our experiments, angiotensin II dose- and time-dependently reduced NO availability, as evaluated by assessing NOx concentrations in culture media (Figure 4A). Previous inhibition of AT1 but not of AT2 receptors prevented angiotensin II–induced changes in NO production by cultured HUVECs (Figure 4B). SPH partially counteracted the angiotensin II action on NO production by cultured HUVECs (Figure 4B). Finally, both DPI and NAC completely prevented the effects of angiotensin II on NO availability (Figure 4B).
Angiotensin II–related changes in NOx concentrations in culture media were comparable with those induced by the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (L-NAME) (Figure 4C). L-NAME also reduced HUVEC motility at a similar level as the one achieved after incubation with angiotensin II (Figure 4D). When HUVECs were incubated with L-NAME, angiotensin II failed to additionally affect both NOx concentration in culture media and HUVEC migratory ability (Figures 4C and 4D, respectively). The NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10−7 mol/L), which is known to stimulate endothelial cell motility,23 completely reversed L-NAME–related changes in NOx concentrations (Figure 4C). In contrast, SNAP was unable to counteract the angiotensin II effect on NO availability (Figure 4C). These data suggest that the angiotensin II actions on NO availability were attributable to increased NO inactivation rather than to reduced NO production. In keeping with this, SNAP also completely blocked the effect of l-NAME on HUVEC motility (Figure 4D) while failing to blunt the inhibitory action exerted by angiotensin II on HUVEC migratory ability (Figure 4D). However, when the SNAP dose was increased (2×10−6 mol/L) to obtain a significant rise in NOx concentration (after 18 hours, 94.7±3.5% of control; NS versus control and P<0.0001 versus angiotensin II 10−7 mol/L, respectively) in angiotensin II–treated cells, a concomitant significant increase in HUVEC motility was observed (after 18 hours, 91.3±3.8% of control; P<0.02 versus control and P<0.0001 versus angiotensin II 10−7 mol/L, respectively). Taken together, these data suggest that angiotensin II reduced NO availability by acting on an oxidant-sensitive pathway. In turn, the reduced NO availability was responsible for the inhibitory action exerted by angiotensin II on HUVEC motility.
Effects of Angiotensin II on HUVEC Proliferation
Using a colorimetric assay for cell proliferation, angiotensin II at 3 different concentrations (10−9, 10−7, and −6 mol/L) had no effects on HUVEC proliferation (after 18 hours of incubation, +1.1±1.9%, +1.5±0.7%, and +2.6±2.3%, respectively; NS versus control), thus excluding a spurious effect of stimulated proliferation on the evaluation of HUVEC migration.
The principal effector of the renin-angiotensin-aldosterone system angiotensin II, either generated in plasma or locally formed by tissue angiotensin-converting enzyme, has been claimed to induce several detrimental endothelial effects.5 Different intracellular pathways can be activated by angiotensin II and lead to vascular endothelial damage. In particular, one of the most important mechanisms involved in angiotensin II–related vascular endothelial cell toxicity is the increased generation of ·O2−,8 which, in turn, is attributable to the AT1-dependent activation of membrane NAD(P)H oxidase.8,9⇓ Once produced, ·O2− acts as a key determinant in altering vascular endothelial cell redox status. Indeed, ·O2− facilitates or induces the formation of other oxidants, such as H2O2, and quenches NO to produce peroxynitrite, ie, one of the most potent intracellular oxidants.8,11,24⇓⇓ In the vascular endothelial cell, the unbalance between NO and ·O2− productions impairs endothelium-dependent vasodilation,25 increases smooth muscle cell proliferation,26 inhibits fibrinolysis,27 activates prothrombotic pathways,27 upregulates redox-sensitive genes codifying for the expression of chemoattractant proteins and adhesion molecules,28 and decreases the inactivation of vasotoxic agents such as homocysteine.29 The present study indicates a novel vascular action mediated by the angiotensin II–related increment in oxidative stress, ie, the inhibition of vascular endothelial cell migration.
In our experiments, angiotensin II reduced HUVEC migration in dose- and time-dependent manners. The Boyden chamber system cannot exclude whether angiotensin II–related changes in HUVEC proliferation contributed to the observed modifications in HUVEC motility. Thus, we analyzed the effects of various concentrations of angiotensin II on HUVEC proliferation. This latter was not affected by angiotensin II over 18 hours of incubation, independently of the tested concentration. Thus, our results strongly suggest that angiotensin II was critical for inhibiting HUVEC migration rather than stimulating HUVEC proliferation.
The inhibitory effect of angiotensin II on HUVEC migration was attenuated by candesartan cilexetil, a selective inhibitor of the AT1 receptor, but not by PD123319, a selective inhibitor of the AT2 receptor. As candesartan cilexetil, both the flavoprotein inhibitor DPI and the antioxidant NAC increased spontaneous HUVEC migration and attenuated the antimigratory effect of angiotensin II. DPI was unable to additionally increase the inhibitory action of candesartan cilexetil on the antimigratory action of angiotensin II, thereby suggesting that an AT1-mediated activation of endothelial NAD(P)H or similar flavoprotein-dependent oxidase was almost solely responsible for the actions exerted by angiotensin II on oxidant generation by HUVECs and HUVEC motility.
In contrast to the other tested inhibitors and to the antioxidant NAC, SPH incompletely blocked the antimigratory action exerted by angiotensin II and did not potentiate the effects of candesartan, DPI, or NAC. In addition, SPH incompletely inhibited the effects of angiotensin II on intracellular oxidative stress and GSSG/GSH ratio as well as NO generation by cultured HUVECs. In contrast to our data, PLD has been recently suggested to be the enzyme responsible for AT1-dependent NAD(P)H oxidase activation in vascular smooth muscle cells.10 However, recent elegant experiments by Touyz and Schiffrin30 indicate that PLD cooperates with other intracellular enzymes, such as protein kinase C, to activate NAD(P)H oxidase also in vascular smooth muscle cells. Accordingly, replication of such experiments in HUVECs demonstrated that coincubation with a selective inhibitor of protein kinase C plus sphinganine completely abolished the increase in oxidative stress attributable to angiotensin II (G. Desideri, unpublished data, 2002). Therefore, PLD seems to be important but not solely responsible for the angiotensin II–related increment of oxidant generation by cultured HUVECs.
The intracellular pathway responsible for the inhibitory action exerted by angiotensin II on HUVEC motility also involved NO availability. Angiotensin II time- and dose-dependently reduced NOx concentrations in culture media, suggesting that a reduction in NO availability did occur in cultured HUVECs after angiotensin II stimulation. Because endogenous NO plays a fundamental role in regulating vascular endothelial cell migratory ability,21 it is intriguing to speculate that angiotensin II inhibited HUVEC motility by reducing NO availability. According to such interpretation, the effects of angiotensin II on NO availability as well as on HUVEC motility were comparable with those induced by the NO synthase inhibitor L-NAME. In addition, when HUVECs were incubated with L-NAME, angiotensin II failed to additionally affect both NO generation by cultured HUVECs and HUVEC migratory ability. These data strongly support the existence of a causal link between the actions of angiotensin on NO availability and HUVEC motility. Of note, the NO donor SNAP, which is known to stimulate endothelial cell migration,20 did not affect angiotensin II–induced changes in NO availability and endothelial cell migration. By contrast, SNAP restored NO availability and completely abolished the antimigratory effect of L-NAME on endothelial cells. However, when the SNAP dose was increased to obtain a significant rise in NOx concentration in angiotensin II–treated cells, a concomitant significant increase in HUVEC motility was observed. These findings suggest that the angiotensin II–related decrement in NO availability was attributable to increased NO breakdown rather than reduced NO production. In this context, it is interesting to observe that the antimigratory action exerted by angiotensin II was strictly dependent on its pro-oxidant capability. Indeed, angiotensin II simultaneously inhibited HUVEC migration and deeply altered the cell redox status, with an increment in intracellular oxidative stress and a decrement in intracellular reduced GSH content. Because ROS are able to inactivate NO,22 it is intriguing to speculate that angiotensin II inhibited HUVEC motility by increasing ROS production and thereby favoring NO breakdown.22 According to this, the antioxidant NAC restored NO availability and attenuated the effect of angiotensin II on HUVEC motility. Interestingly, a ROS-related reduction in NO availability also represents the main mechanism underlying the inhibitory action exerted by CD40L, ie, a peptide that is deeply involved in the pathobiology of vascular diseases, on vascular endothelial cell motility.31
Migration assays by the widely used Boyden chamber system21 may be affected by cellular events that are only indirectly related to cell migration. Angiotensin II may induce rearrangements of the cytoskeleton, alterations in cell adhesion or spreading, or change of cell size or shape. All these phenomena could mimic an apparent HUVEC migration through the polycarbonate membrane. To avoid this bias, we counted endothelial cells under a ×20 magnification, which allowed us to clearly identify each endothelial cell that migrated through the pores of the inserted membrane. In addition, we counted endothelial cells that were lost in culture media after migration and failed to find significant differences both in the presence and absence of different concentrations of angiotensin II (data not shown). Therefore, it is extremely unlikely that angiotensin II–related changes in HUVEC size, shape, or adhesion significantly affected our results. In contrast, the small number of HUVECs observed in the lower area of the polycarbonate membrane after angiotensin II incubation clearly reflects an antimigratory action exerted by angiotensin II.
In conclusion, angiotensin II, the main effector of the renin-angiotensin system, is known to play a fundamental role in the regulation of various endothelial functions.5 Endothelial cell migration is a critical event in atherogenesis and vascular response to injury.4 Furthermore, abnormalities in vascular endothelial cell migratory ability have been suggested to be essential in restenosis after percutaneous transluminal coronary angioplasty.4
The present study demonstrates that angiotensin II, either locally formed by cultured HUVECs or added to culture media, inhibits cell migration by reducing NO availability. In addition, our study clearly shows that such angiotensin II–related decrement in NO availability is attributable to an AT1-dependent increment in oxidant generation by cultured HUVECs.
The above actions of angiotensin II are likely to play a physiological role in modulating vascular endothelial cell motility. Nevertheless, the antimigratory properties of angiotensin II have all the potential to be of particular relevance in human pathologic conditions, implying an abnormal vascular endothelial cell response to injury.
- Received March 17, 2003.
- Accepted April 28, 2003.
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- ↵Desideri G, Ferri C, Bellini C, De Mattia G, Santucci A. Effects of ACE inhibition on spontaneous and insulin-stimulated endothelin-1 secretion. Diabetes. 1997; 46: 81–86.
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- ↵Murohara T, Witzenbichler B, Spyridopoulos I, Asahara T, Ding B, Sullivan A, Losordo DW, Isner JM. Role of endothelial nitric oxide synthase in endothelial cell migration. Arterioscler Thromb Vasc Biol. 1999; 19: 1156–1161.
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- ↵Pueyo ME, Gonzales W, Nicoletti A, Savoie F, Arnal JF, Michel JB. Angiotensin II stimulates endothelial vascular cell adhesion molecule-1 via nuclear factor-κB activation induced by intracellular oxidative stress. Arterioscler Thromb Vasc Biol. 2000; 20: 645–651.
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