Angiotensin II Induces Capillary Formation From Endothelial Cells Via the LOX-1–Dependent Redox-Sensitive Pathway
Angiotensin II (Ang II) induces angiogenesis by stimulating reactive oxygen species–dependent vascular endothelial growth factor (VEGF) expression. Ang II via type 1 receptor upregulates the expression of LOX-1, a lectin-like receptor for oxidized low-density lipoprotein. LOX-1 activation, in turn, upregulates Ang II type 1 receptor expression. We postulated that interruption of the feedback loop between Ang II and LOX-1 might attenuate Ang II–induced VEGF expression and capillary formation. In vitro experiments showed that Ang II (1 nmol/L) induced the expression of LOX-1 and VEGF and enhanced capillary formation from human coronary endothelial cells in Matrigel assay. Ang II–mediated expression of LOX-1 and VEGF, capillary formation, intracellular reactive oxygen species generation, and phosphorylation of p38 as well as p44/42 mitogen-activated protein kinases, were suppressed by anti–LOX-1 antibody, nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin and the Ang II type 1 receptor blocker losartan, but not by the Ang II type 2 receptor blocker PD123319. Expression of VEGF and capillary formation induced by Ang II were also inhibited by the p44/42 mitogen-activated protein kinase inhibitor U0126 and the p38 mitogen-activated protein kinase inhibitor SB203580. In ex vivo experiments, Ang II stimulated capillary sprouting from aortic rings from wild-type mice, and this phenomenon was significantly attenuated by pretreatment of aortic rings with anti–LOX-1 antibody, apocynin, and losartan, but not by PD123319. Importantly, Ang II–induced capillary sprouting was minimal from aortic rings from LOX-1 null mice compared with wild-type mice. These findings suggest that small concentrations of Ang II promote capillary formation by inducing the expression of VEGF via Ang II type 1 receptor/LOX-1–mediated stimulation of the reactive oxygen species-mitogen-activated protein kinase pathway.
Angiotensin II (Ang II) has been implicated in both angiogenesis and pathological vascular growth.1 The contributory role of Ang II in angiogenesis is supported by several lines of evidence1,2: (1) Ang II type 1 receptor (AT1R) stimulation promotes endothelial cells proliferation; (2) Ang II induces capillary formation from endothelial cells by inducing the expression of vascular endothelial growth factor (VEGF) via nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase/reactive oxygen species (ROS) pathway; (3) ischemia-induced angiogenesis is mediated through AT1R-mediated upregulation of VEGF and its receptor; (4) AT1R antagonists and angiotensin-converting enzyme inhibitors inhibit tumor-associated angiogenesis in murine models; (5) VEGF-mediated angiogenesis is impaired by AT1R blockade in cardiomyopathic hamster hearts; and (6) coronary capillary angiogenesis is mediated via AT1R-mediated upregulation of VEGF at the insulin-resistant stage in the noninsulin-dependent diabetes mellitus rat model.
LOX-1, a lectin-like scavenger receptor for oxidized low-density lipoprotein (LDL), is expressed primarily on endothelial cells.3 This receptor is upregulated by Ang II.4 In turn, activation of LOX-1 upregulates AT1R expression.5 Activation of both AT1R and LOX-1 induces intense oxidative stress.3 We postulated that interruption of this positive feedback loop between LOX-1 and AT1R activation might attenuate Ang II–induced angiogenesis. This concept was examined in 2 different models of capillary growth, first, an in vitro model of capillary formation in human coronary artery endothelial cells (HCAECs) and, second, an ex vivo model of capillary sprouting from mouse aortic rings.
Materials and Reagents
Monoclonal antibody against human LOX-1 raised in mice with the human Fc portion of IgG and monoclonal antibody against mice LOX-1 raised in rats with the mouse Fc portion of IgG have been reported earlier to block the effect of LOX-1.6,7 The following reagents and antibodies were purchased: Matrigel with reduced growth factor (BD Biosciences); the AT1R blocker losartan, the Ang II type 2 receptor (AT2R) blocker PD123319, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, and the p44/42 MAPK inhibitor U0126 (Sigma); the NADPH oxidase inhibitor apocynin (Aldrich); 2′,7′-dichlorodihydrofluorescein diacetate (Cayman); human or mouse IgG; and all of the primary antibodies for Western blot analysis except anti–LOX-1 antibody (Santa Cruz Biotechnology). U0126, SB203580, and apocynin were dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide was <0.1% in these experiments.
In Vitro Studies
HCAECs were originally purchased from Clonetics and cultured at 37°C under 5% CO2 in endothelial cell basal medium–2 (Clonetics) supplemented with 5% FBS, penicillin/streptomycin, and endothelial cell growth supplement.5,6 Fourth- to sixth-generation HCAECs were used in this study. In some experiments, HCAECs were supplemented with 5% FBS but without endothelial cell growth supplement.
Capillary Tube Formation
Matrigel was thawed on ice overnight and spread evenly over each well (30 μL) of a 24-well plate. The plates were incubated for 1 hour at 37°C to allow Matrigel to polymerize. HCAECs were seeded at 3×104 per well and grown in 500 μL of endothelial cell basal medium–2 supplemented with 5% FBS and without endothelial cell growth supplement for 24 hours in a humidified 37°C, 5%-CO2 incubator. In some experiments, endothelial cells were cultured in the presence or absence of different chemicals or antibodies. After washing, plates were fixed using 70% ice-cold ethanol. Capillary formation was visualized by staining with hematoxylin and eosin and assessed as described previously.8
HCAECs were exposed to Ang II (0, 0.1, 1, 5, 10, 20, 50, and 100 nmol/L) for 24 hours. In parallel studies, HCAECs were pretreated for 30 minutes with losartan (1, 2, 5, and 10 μmol/L), PD123319 (10 μmol/L), anti–LOX-1 antibody (10 μg/mL), nonspecific IgG (10 μg/mL), apocynin (600 μmol/L), U0126 (10 μmol/L), SB203580 (10 μmol/L), or dimethyl sulfoxide (as vehicle control) before exposure to Ang II. These concentrations and durations of incubation were chosen on the basis of published data9–11 and modified based on pilot experiments.
Measurement of Intracellular ROS
Intracellular ROS was measured with the fluorescent signal 2′,7′-dichlorodihydrofluorescein diacetate.11 HCAECs cultured in 2-well chamber slides were incubated with 10 μmol/L of 2′,7′-dichlorodihydrofluorescein diacetate in PBS for 30 minutes. 2′,7′-Dichlorodihydrofluorescein diacetate is nonfluorescent until the acetate groups are removed by intracellular ROS. The ROS-mediated fluorescence was observed under a fluorescent microscope (Nikon) with excitation set at 502 nm and emission set at 523 nm.
Western Blot Analysis
Cell protein was extracted for the expression analysis of LOX-1, p38 and p44/42 MAPKs, VEGF, and β-actin using standard Western blot methodologies.6 Densities of protein bands relative to β-actin were analyzed.
Ex Vivo Studies
Capillary Sprouting From Aortic Rings
Thoracic aortas were isolated from 8-week-old male C57BL/6 mice (Jackson Laboratories) and LOX-1 null mice anesthetized with pentobarbital sodium (80 mg/kg, IP), cut into 1-mm-thick sections, and embedded in 24-well Matrigel-coated plates. DMEM supplemented with 5% FBS, 20 U/mL of heparin (Sigma), and penicillin/streptomycin was added to each well of gelled Matrigel. The number and length of microvasculature sprouting from each aortic ring were assessed as described previously.12,13 This study conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All of the experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Usage Committee. The LOX-1 null mice were developed in our laboratory, and their generation has been described recently.7
Data are expressed as mean±SEM. All of the values were analyzed by using 1-way ANOVA and the Newman-Keuls-Student t test. The significance level was chosen as P<0.05.
Ang II Induces Capillary Tube Formation From Endothelial Cells Via the AT1R/LOX-1 Pathway
As shown in Figure 1A and 1B, low concentrations of Ang II (0.1, 1, 5, 10, and 20 nmol/L) led to the formation of capillary-like structures. The maximum capillary formation occurred in response to 1 nmol/L concentration of Ang II. In contrast, higher concentrations of Ang II (50 and 100 nmol/L) were noted to cause less tube formation. Based on these findings, we chose the most proangiogenic concentration of Ang II (1 nmol/L) in subsequent experiments.
Biological effects of Ang II are mediated by activation of Ang II receptors, of which 2 major subtypes, AT1R and AT2R, have been identified.2 Most of the cardiovascular actions of Ang II have been attributed to AT1R activation.2 We observed that pretreatment of HCAECs with the AT1R blocker losartan (1, 2, 5, and 10 μmol/L) suppressed Ang II–induced capillary tube formation in a dose-dependent manner (Figure 1C). In contrast, pretreatment with the AT2R blocker PD123319 (10 μmol/L) had no effect on Ang II–induced capillary tube formation. Losartan (10 μmol/L) and PD123319 (10 μmol/L) alone also had no effect on capillary formation.
Ang II upregulates LOX-1 expression in an NADPH oxidase–dependent manner.3–5 In keeping with the published data, we observed that Ang II upregulated LOX-1 expression in HCAECs (Figure 2A). Pretreatment with losartan (10 μmol/L) or the specific NADPH oxidase inhibitor apocynin (600 μmol/L) blocked Ang II–mediated LOX-1 expression. In keeping with previous studies,14 pretreatment with anti–LOX-1 antibody (10 μg/mL) reduced Ang II–mediated LOX-1 expression. Importantly, nonspecific IgG had no effect on LOX-1 expression (Figure 2A).
Pretreatment with losartan, apocynin, and anti–LOX-1 antibody, but not nonspecific IgG, markedly suppressed capillary tube formation induced by Ang II (1 nmol/L; Figure 2B). Losartan, apocynin, or anti–LOX-1 antibody alone had no effect on Ang II–mediated capillary tube formation (data not shown).
Ang II and Redox-Sensitive Signaling Events
Ang II is a potent stimulus for expression and activation of vascular NADPH oxidases.1,2 NADPH oxidases are a major source of ROS generation in endothelial cells.1,2 ROS upregulates LOX-1 expression, and LOX-1 activation itself can stimulate ROS formation.3 We, therefore, measured intracellular ROS generation in HCAECs. As shown in Figure 3A, pretreatment of HCAECs with losartan, apocynin, or anti–LOX-1 antibody markedly reduced the Ang II–induced increase in dichlorofluorescein fluorescence, reflecting a reduction in intracellular ROS generation. Nonspecific IgG had no effect on ROS generation induced by Ang II. Losartan, apocynin, or anti–LOX-1 antibody alone had no effect on basal ROS generation (data not shown).
It has been well documented that many angiogenesis-related responses are redox sensitive1 and that AT1R/LOX-1 activation in endothelial cells initiates a cascade of redox-sensitive signaling events, including activation of the MAPK pathway.1,3,5,9,10 Accordingly, we determined the expression and activation of p38 and p44/42 components of MAPKs. As shown in Figure 3B, expression of p38 as well as p44/42 MAPKs was unaffected, but their phosphorylation was enhanced by treatment of HCAECs with Ang II. Pretreatment with losartan, anti–LOX-1 antibody, or apocynin blocked Ang II–mediated phosphorylation of p38 and p44/42 MAPKs. As a control, nonspecific IgG had no effect on the phosphorylation of p38 and p44/42 MAPKs. Losartan, apocynin, or anti–LOX-1 antibody alone had no effect on the basal expression or activation of p38 and p44/42 MAPKs (data not shown).
Ang II induces angiogenesis by AT1R-mediated upregulation of VEGF in a redox-sensitive manner.1 In keeping with these data, we observed that VEGF was induced by Ang II, and its upregulation was inhibited by pretreatment with losartan or apocynin (Figure 3B). Importantly, we found that anti–LOX-1 antibody markedly inhibited Ang II–induced VEGF expression. Nonspecific IgG had no effect on the expression of VEGF in response to Ang II. Furthermore, anti–LOX-1 antibody, losartan, or apocynin alone had no effect on the basal expression of VEGF (data not shown).
As shown in Figure 4A, Ang II–induced VEGF expression was also inhibited by the specific p44/42 MAPK inhibitor U0126, as well as the p38 MAPK inhibitor SB203580. U0126 and SB203580 had no effect on LOX-1 expression induced by Ang II, indicating that MAPK signaling is an event downstream of LOX-1 expression. U0126 and SB203580 also had no effect on the basal expression of VEGF (data not shown).
Involvement of the MAPK Pathway in Ang II–Induced Capillary Tube Formation
To examine whether the MAPK pathway is involved in Ang II–induced capillary tube formation, we used specific MAPK inhibitors. As shown in Figure 4B, capillary tube formation induced by Ang II was dramatically suppressed in the presence of the p44/42MAPK inhibitor U0126 or the p38 MAPK inhibitor SB203580. U0126 or SB203580 alone had no effect on tube formation (data not shown).
Ang II Induces Capillary Sprouting From the Aortic Ring Via the AT1R/LOX-1/NADPH Oxidase Pathway
In keeping with the previous data,12,13 mouse aortic ring explants embedded in Matrigel gave rise to capillary-like structures. Treatment of aortic rings from wild-type mice with Ang II (1 nmol/L) for 7 days markedly enhanced capillary sprouting, as shown by the significant increase in the number and length of newly sprouted vessels. The length and number of newly sprouted vessels both were reduced by ≈70% by pretreatment with losartan, apocynin, or anti–LOX-1 antibody but did not change in the presence of PD123319 in this ex vivo assay (Figure 5). Losartan, apocynin, or anti–LOX-1 antibody alone also had no effect on microvascular sprouting in basal state (data not shown). To further confirm the role of LOX-1 in Ang II–mediated capillary formation, we examined capillary sprouting from aortic rings from LOX-1 null mice and found that deletion of LOX-1 markedly inhibited Ang II–induced capillary formation (Figure 6).
This study confirms previous observations15–17 that Ang II in small amounts induces capillary tube formation from vascular endothelial cells. In keeping with previous observations, we show that the proangiogenic effects of Ang II are mediated by AT1R activation. We confirmed this phenomenon in 2 independent models, first, an in vitro model based on HCAECs and, second, an ex vivo model of capillary sprouting from mouse aortic rings.
The AT2R blocker PD123319 in our studies had no effect on capillary tube formation from HCAECs or mouse aortic rings. Previous studies have shown variable effects of AT2R on angiogenic response to Ang II.15–18 It has been suggested that AT2R activation has effects opposite to those of AT1R activation.19 The antiangiogenic effect of AT2R activation has been confirmed not only by the use of chemical blockers but also by the use of AT2R null mice.15,18,20
It has been amply demonstrated that Ang II is a potent stimulus for ROS generation, and ROS release is probably the basis of capillary formation.1 ROS are generated primarily as a result of NADPH oxidase activation.1,10,11 In the present study, we demonstrate that Ang II induces ROS generation in endothelial cells with subsequent activation of both p38 and p44/42 MAPKs. The role of ROS generation and MAPK activation in capillary tube formation became evident in experiments wherein specific inhibitors of NADPH oxidases and MAPKs blocked the angiogenic response to Ang II. This study also demonstrates that Ang II enhances the expression of VEGF, which is a consequence of NADPH oxidase–mediated ROS generation and subsequent activation of redox-sensitive pathways. Although ROS are necessary for angiogenesis, excessive concentrations of ROS can induce cell injury and block generation of new capillaries.1 This phenomenon was confirmed in the present study as well (Figure 1).
It has been demonstrated that LOX-1 is upregulated by oxidized LDL, shear stress, proinflammatory cytokines, and Ang II.3 Upregulation of LOX-1 expression by Ang II is mediated by AT1R activation, because AT1R, but not the AT2R, blockers attenuate this effect.21 In other studies, we showed that oxidized LDL via LOX-1 upregulates AT1R expression in endothelial cells.3,5 We postulated that the 2 major mediators of atherogenesis, Ang II and oxidized LDL, exert their effect through the AT1R/LOX-1 pathway.3 Activation of both AT1R and LOX-1 enhances oxidant stress, and it is likely that the activation of redox-sensitive pathways leads to capillary formation and atherogenesis.3 These concepts were supported by studies in hypercholesterolemic rabbits and apolipoprotein-E–null mice that exhibit high levels of LOX-1 and AT1R.22,23 Most importantly, treatment of animals with AT1R blockers dramatically reduced LOX-1 expression and atherogenesis.22,23 It is of note that the atherosclerotic regions, particularly in humans, are rich in microvasculature.24 Formation of new capillaries may well be proatherogenic by providing nutrition to the growing plaque and facilitating the migration of microphages while the plaque is in the growth phase.
We postulated that Ang II may enhance VEGF expression and capillary formation via LOX-1 upregulation. Indeed, our study demonstrates that this is the case. In 2 different models of capillary tube formation, we showed that the anti–LOX-1 antibody suppressed the angiogenic response to Ang II. This was not an incidental finding, because nonspecific IgG did not have a similar effect. Studies with the use of aortic rings from the LOX-1 null mouse revealed a minimal angiogenic response to Ang II. Both losartan and apocynin blocked Ang II–mediated LOX-1 expression, as well as capillary tube formation. It is noteworthy that the activation of MAPKs was found to be downstream of LOX-1 expression, because the inhibitors of MAPKs did not influence the expression of LOX-1, yet they inhibited capillary tube formation.
This study demonstrates the potent angiogenic response to Ang II, which is mediated in large part by the induction of LOX-1 via AT1R and the generation of ROS. We have shown recently the significance of LOX-1 in LDL receptor/LOX-1 double-null mice that are resistant to atherogenesis despite the feeding of an atherogenic diet.7 The current study further suggests that LOX-1 may be a reasonable target of antiatherogenesis therapy.
Sources of Funding
This study was supported in part by funds from the Department of Veterans Affairs.
The first 2 authors contributed equally to this work.
- Received June 14, 2007.
- Revision received July 16, 2007.
- Accepted August 29, 2007.
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