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* Research Laboratories of Schering AG, Experimental Oncology, 13342 Berlin, Germany; and
# Department of Cardiology and Pneumology and
Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Freie Universität Berlin, 12200 Berlin, Germany
1Correspondence: Benjamin Franklin Medical Center, Department of Cardiology and Pneumology, Hindenburgdamm 30 D-12200 Berlin, Germany. E-mail: thomas.walther{at}ukbf.fu-berlin.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: alginate implant angiogenesis • angiotensin receptor AT1 • angiotensin receptor AT2
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Meanwhile, several direct stimulators and inhibitors of angiogenesis have been identified; most of them are proteins such as the vascular endothelial growth factor (VEGF-A) (2)
, angiopoietin-1 (3)
, or angiopoietin-2 (3)
and appear to regulate essential regulatory pathways of angiogenesis.
A distinct mechanism regulating endothelial cell growth has been proposed for the octapeptide angiotensin II (ANG II), the main effector molecule of the renin-angiotensin system (RAS), thus regulating blood pressure as well as body fluid homeostasis (4)
. On the cellular level, ANG II exerts growth stimulation of quiescent endothelial cells via the AT1 receptor (4)
. In addition to findings in cell culture systems, several in vivo angiogenesis models were used to investigate the effects of ANG II on angiogenesis. Most in vivo model systems were based on a bolus application or a sustained release of high amounts of exogenous ANG II from locally implanted pellets or sponge material. Thereby, an induction of angiogenesis in the chorioallantoic membrane (CAM) assay (5)
, the corneal pocket model of the rabbit (6)
, and in the rat subcutaneous sponge granuloma model (7)
was shown. These in vivo model systems demonstrated the effect of ANG II on the quiescent endothelial cell, but not the effect on growing endothelial cells stimulated by specific endothelial cell growth factors.
In the presence of angiogenic growth factors such as bFGF or VEGF, however, ANG II may inhibit growth of endothelial cells. Previous data have suggested the AT2 receptor as a mediator of this effect (4)
. Although earlier in vivo experiments failed to provide evidence for a functional implication of the AT2 receptor, abundant expression of AT2 receptors in fetal tissues (8)
, the endometrium (9)
, and wound healing tissue (10)
as well as the attenuation of neointima formation by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery (11)
propose a functional role of the AT2 receptor subtype whose physiological role is not yet clear. Nevertheless, speculations on an anti-angiogenic potential of endogenous ANG II acting on AT2 receptors are in obvious conflict with reports on the regular embryonic growth of AT2 receptor knockout mice (12)
.
While the role of both ANG II receptors in the process of in vivo angiogenesis is unclear and their interaction has to be clarified, the inhibitory effect of endogenous ANG II on angiogenesis is well established. Previous reports showed an increased density of myocardial capillaries in spontaneously hypertensive rats (SHR) by ANG II withdrawal applying ACE inhibitors (13)
. Another line of evidence for an inhibitory action of ANG II on growth factor-driven angiogenesis was obtained in a rabbit model of hindlimb ischemia. Treatment with the ACE inhibitor enalapril, which decreases circulating levels of ANG II, led to an increase of angiogenesis comparable to the effect of VEGF (14)
.
To investigate the hypothetical role of ANG II as an endogenous receptor-based inhibitor of angiogenesis, we applied the alginate tumor angiogenesis model (15)
. This model system provides several advantages over previous experimental strategies that used high amounts of locally applied ANG II. 1) Studies by Schirner et al. (16)
using two human tumor xenograft in mice with different growth kinetics showed that angiogenesis measurement based on the alginate model in mice correlate with measurements of the microvessel density (MVD) in solid tumors, which is the "gold standard" in the clinical practice. 2) In vivo angiogenesis is induced by angiogenic growth factors continuously released by tumor cells entrapped in alginate beads (15
, 17)
. As tumor cells and hypoxic epithelial cells release a similar pattern of angiogenic growth factors (18)
, our model system may also be representative of the neo-vascularization process in ischemic tissues. 3) Induction of angiogenesis is assessed by the high molecular weight blood pool marker FITC-dextran enabling sensitive and reliable determination of functional blood volume at alginate implants (15)
. 4) The entire angiogenesis process is exposed to the endogenous level of ANG II which can be easily affected by established therapies or genetical manipulations of the endogenous ANG II level or ANG II receptors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Sodium alginate of low viscosity was dissolved in sterile saline to a final concentration of 1.5%. Tumor cells were harvested from cell culture at 60–80% confluence. After centrifugation, the tumor cell pellet was directly resuspended with the alginate solution to the desired cell number and thereafter filled into a reservoir. Droplets containing tumor cells were produced by extrusion of the alginate solution through a 12 gauge cannula. The tumor cell alginate solution was dropped into a swirling bath of 80 mM CaCl2. The calcium ions caused immediate gelling of each droplet by an exchange of sodium from the alginate. The size of the beads was minimized by a laminar air flow along the cannula. After incubation in the CaCl2 bath for a further 30 min, the beads were washed twice with buffer, centrifuged, and prepared for injection. All procedures were carried out under sterile conditions at 37°C.
Alginate in vivo angiogenesis assay
Female Swiss nu/nu mice, male C57Bl/6 mice, male NMRI mice, male transgenic NMRI mice of the strain TGM(rAOGEN)123 (19)
, or male AT2 receptor subtype knockout mice (20)
weighing 
22 g were injected s.c. with 0.1 mL alginate beads containing tumor cells into the inguinal mammary fad pad. Because of the size of the inoculation, it was not possible to implant the beads directly into the mammary gland. However, the approach used here is also considered to be an orthotopic implantation. Control mice were implanted with 0.1 mL alginate beads without tumor cells. Each alginate implant consists of 50 alginate beads. The alginate matrix is diffusible for angiogenic growth factors. Capillaries grow into the space between the beads but do not invade the beads themselves. FITC-dextran with an average molecular weight of 150,000 was dissolved in saline to a final concentration of 1%. At the end of the experiment, 0.2 mL of 1% FITC-dextran solution (100 mg/kg) was i.v. injected into the lateral tail vein of mice. Alginate implants were rapidly removed 20 min after FITC-dextran injection, weighed, and transferred to tubes containing 2 mL saline. The tubes were mixed by vortexing for 20 s and centrifuged (3 min, 1000 g). After dilution (1:1), fluorescence of the supernatant was measured with a fluorescence spectrophotometer by exiting at 492 nm and emission at 515 nm. Calibration curves were prepared in saline (0.01–10 µg/mL FITC-dextran). After removal of alginate implants from the animals, all procedures were carried out in the dark.
Treatment protocols
Alginate implant-bearing mice were treated with the ACE inhibitor enalapril (0.1–1 mg/kg), the ACE inhibitor captopril (1–0 mg/kg), the AT1 receptor subtype antagonist losartan (10 mg/kg), the AT2 receptor subtype antagonist PD 123319 (3 mg/kg), the bradykinin receptor antagonist Hoe 140 (20 mg/kg), or the blood pressure-lowering drug dihydralazine (5 mg/kg). All compounds were daily applied by s.c. injection at the treatment intervals indicated.
RT-PCR analysis of AT1 receptors and AT2 receptors
Total cellular RNA was extracted from confluent cultures of LL2 and MCF-7 cells using the RNeasyTM protocol (Qiagen, Hilden, Germany). As the chosen AT receptor primers do not span intervening genomic introns, 5 µg of each RNA was digested with RNase-free DNase I (Roche, Mannheim, Germany) before reverse transcription. Reverse transcription was performed using SuperscriptTM reverse transcriptase (Invitrogen, Eggenstein, Germany) and random hexamer primers (Roche) according to the manufacturer’s recommendations. PCR reactions were carried out using Taq DNA polymerase (Invitrogen). Primer synthesis (TIB MOLBIOL, Berlin, Germany) was based on published human and murine AT receptor cDNA sequences (21)
, respectively, and were as follows: AT1 sense, 5'-AGA ATC CAA GAT GAT TGT CC; AT1 antisense, 5'-TTG GCT ACA AGC ATT GTG C; AT2 sense, 5'-TCT TTG GAC CTG TGA TGT GC; AT2 antisense, 5'-CTG CCA TCT TCA GGA CTT GG. Cycling conditions were as follows: 94°C, 1 min; 55°C, 1 min; 72°C, 45 s. To correct for nucleotide mismatches of AT1 primers with LL2 cDNA, the initial five cycles of the AT1 receptor PCR were performed at annealing temperatures of 45°C. As additional positive controls, cDNA synthesized from human kidney (AT1 receptor) poly-A RNA (Clontech, Heidelberg, Germany), and human fetal heart (AT2 receptor) poly-A RNA (Clontech) were amplified. A total of 35 cycles was carried out and amplified products were resolved on agarose gels containing ethidium bromide and documented using a GDS 5000 video gel documentation system (Ultraviolet Products, Cambridge, UK).
Statistics
All data are expressed as means ± SE and were analyzed by Student's t test. P values <0.05 were accepted as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). A similar increase of angiogenesis by enalapril was observed in nude mice inoculated with alginate beads encapsulating human MCF-7 mammary carcinoma cells. Treatment of mice with 1.0 mg/kg enalapril exerted an increase of angiogenesis by 127% (Fig. 1B
). A similar increase of angiogenesis was obtained with daily doses of captopril within the dose range of 1 to 10 mg·kg-1·day-1 (data not shown).
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Further investigations were performed to determine the time course of angiogenesis induction by enalapril. Investigation of different treatment intervals showed a period of 11 days to be necessary for stimulation of angiogenesis by enalapril (Fig. 1C
). A treatment period of <11 days remained without a statistically significant effect on angiogenesis of alginate implants.
Additional studies were initiated in order to rule out either a potential contribution of the bradykinin pathway or blood pressure-lowering effects to the stimulatory effect of enalapril on angiogenesis. The bradykinin antagonist Hoe 140 (20 mg·kg-1·day-1; day 0–day 11; s.c.) and the blood pressure-lowering drug dihydralazine (5 mg·kg-1·day-1; day 0–day 11; s.c.) were without effect on induction of angiogenesis (data not shown). Taken together, these data strongly indicate that endogenous ANG II at normal plasma levels exerts suppression of growth factor-driven in vivo angiogenesis.
Inhibition of angiogenesis by ANG II is receptor-specific
To elucidate which angiotensin receptor mediates anti-angiogenic effects of ANG II, specific receptor antagonists were investigated: the AT1 receptor antagonist losartan and the AT2 receptor antagonist PD 123319 (22)
. Treatment with losartan was performed by daily s.c. administration of 10 mg/kg for 11 days starting on the day of alginate implantation. Treatment of mice bearing MCF-7 alginate implants showed an induction of angiogenesis by 220% over controls (Fig. 2
). In contrast to the effect of the AT1 subtype receptor blockade, daily s.c. administration of the AT2 receptor antagonist PD 123319 failed to affect angiogenesis of alginate implants encapsulating human MCF-7 carcinoma cells (Fig. 2)
. These results suggest that normal levels of ANG II exert anti-angiogenic effects via binding to and activation of the AT1 receptor subtype. The combination of both ANG II subtype receptor antagonists revealed a stimulation of in vivo angiogenesis identical to that obtained with losartan alone (Fig. 2)
. This finding demonstrates that withdrawal of ANG II by the ACE inhibitor enalapril mainly affects the AT1 subtype receptor pathway.
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Elevated ANG II levels stimulate angiogenesis
To further substantiate the anti-angiogenic potential of endogenous ANG II, we investigated the degree of angiogenesis induction after implantation of alginate beads into transgenic NMRI mice of the strain TGM(rAOGEN)123. These mice are transgenic for rat angiotensinogen, the precursor molecule of ANG II, which results in a fourfold increased level of plasma angiotensinogen and plasma ANG II (19)
. The preceding experiments showed an identical stimulation of angiogenesis in control NMRI mice with alginate implants encapsulating LL2 cells in comparison to C57Bl/6 mice. Implantation of alginate beads encapsulating mouse LL2 carcinoma cells into TGM(rAOGEN)123 mice led to an unexpected stimulation of angiogenesis by 200% compared with controls (Fig. 3
). This stimulation remained unaffected by the AT1 subtype receptor antagonist losartan, but was abolished by the AT2 subtype receptor PD123319. This suggests that elevated ANG II may have a stimulatory action on in vivo angiogenesis independent of its effects on the AT1 subtype receptor. The blockade of the angiogenesis stimulation by PD123319 implies an involvement of the AT2 subtype receptor.
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Inhibition of angiogenesis in AT2 subtype receptor knockout mice
To further substantiate a potential role for the AT2 subtype receptor but circumvent the potential partial agonism of the available AT2 receptor blockers, we used AT2 subtype receptor knockout mice. These mice were injected with alginate implants encapsulating mouse LL2 carcinoma cells. Similar to previous experiments, the implantation of tumor cell containing alginate implants into mice led to an fourfold increase of angiogenesis compared with controls bearing alginate implants without tumor cells. In contrast, a significantly lower induction of angiogenesis was obtained in AT2 receptor knockout mice, which provides strong evidence that ANG II stimulates angiogenesis through functional AT2 receptors (Fig. 4
).
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The abolished stimulation of angiogenesis in mice that do not express the AT2 subtype receptor could be explained by the absence of a receptor subtype that transmits the putative stimulatory signal by ANG II but also by the dominance of the remaining AT1 receptor, which still exerts anti-angiogenic effects. To address the latter possibility, we treated AT2 receptor knockout mice with the ACE inhibitor enalapril to decrease circulating levels of ANG II. As shown in Fig. 4
, enalapril treatment led to an induction of angiogenesis even above the level of controls with intact AT2 subtype receptor. The degree of stimulation above controls was similar with the degree obtained in experiments shown by Fig. 1A-C
. This finding demonstrates that the impaired angiogenesis in AT2 knockout mice is due to the autonomous action of the inhibitory AT1 receptor subtype.
Analysis of AT1- and AT2 receptor mRNA expression by RT-PCR yielded no specific mRNA transcript for either the AT1 receptor or the AT2 receptor in both tumor cell lines (Fig. 5
). Our results thereby exclude direct effects of ANG II on tumor cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To answer this question, we applied the highly sensitive and reliable alginate angiogenesis model in mice. The angiogenesis process in the alginate model is initiated by several angiogenic growth factors such as VEGF, bFGF, VEGF-C, and angiopoietin-1, which are released by encapsulated tumor cells, and does not rely on application of ANG II for induction of angiogenesis. All investigational manipulations focused on the level of endogenous ANG II or its respective receptors. Furthermore, because tumor cells release a similar pattern of angiogenic growth factors as hypoxic epithelial cells (18)
, we are confident that the alginate angiogenesis model can also be indicative of the angiogenesis process induced by ischemia.
Previous studies by other groups showed that locally high doses of ANG II induce angiogenesis by acting on AT1 subtype receptors (5
6
7)
. However, recent findings and our own data with ACE inhibitors showed that a withdrawal of the endogenous ANG II can stimulate in vivo angiogenesis as well (14
, 22)
. As these finding are in clear conflict with previous studies, we studied the question of whether the AT2 receptor itself can mediate an anti-proliferative signal that becomes unmasked after ANG II withdrawal as has been suggested by other investigators (23)
. Although this hypothesis seems attractive, supporting evidence was obtained mainly from nonendothelial cell culture systems. The major hurdle for further substantiation of the proposed hypothesis remained the lack of defined AT2 receptor antagonists. In the present studies, therefore, we used both the AT2 receptor knockout mice and AT2 receptor antagonist PD123319.
Besides the right transgenic animal and/or pharmacological treatment, the effects on angiogenesis seem to depend strongly on the chosen angiogenesis model. Since our data are opposing actual findings by Silvestre et al., who also used AT2-deficient mice but in a model of surgically induced hindlimb ischemia (24)
, general conclusions made by investigations on one angiogenesis model could go astray. Consequently, none of the published animal angiogenesis models can cover all aspects of the in vivo angiogenesis process because of its complexity.
However, our present findings provide for the first time strong evidence for a functional cross-regulation of in vivo angiogenesis by ANG II involving both AT1 and AT2 subtype receptors with opposing actions. The AT1 subtype receptor exerts an anti-angiogenic effect after stimulation by endogenous ANG II in the alginate tumor angiogenesis model (15)
. Its inhibition by a selective AT1 receptor antagonist or by withdrawal of ANG II using an ACE inhibitor leads to a stimulation of in vivo angiogenesis. Besides the inhibitory AT1 receptor function, an additional stimulatory AT2 receptor function on in vivo angiogenesis was discovered. In mice with normal ANG II levels, this regulatory axis became unmasked only if the AT2 subtype receptor was absent, as achieved in AT2 knockout mice. Despite the presence of a stimulatory AT2 subtype receptor, the inhibitory activity of the AT1 subtype receptor was predominant, as indicated by the stimulatory effect on in vivo angiogenesis after ANG II withdrawal. However, an increase of the endogenous ANG II level as obtained with the transgenic overexpression of the proenzyme angiotensinogen may shift the balance from the inhibitory AT1 receptor axis to the stimulatory AT2. In this case, the AT2 subtype receptor is predominant over the AT1 subtype receptor, which is indicated by the complete blockade of angiogenesis with the AT2 receptor antagonist.
This stimulatory effect can also be concluded from the different action of the experimental AT2 receptor antagonist PD123319. While PD123319 remained without effect on in vivo angiogenesis in mice with normal levels of ANG II, an inhibition of the angiogenesis induction was obtained in mice with elevated ANG II levels. Investigation in AT2 knockout mice confirmed the proangiogenic action of the AT2 receptor and ruled out doubts on a potential partial agonism of PD123319 in doses used in the present study (25)
.
It is important to note that the main function of ANG II is to maintain body fluid homeostasis in response to a drop of the perfusion pressure through an AT1 receptor-mediated vasoconstriction. We showed a novel and distinct biological action of Ang II that is an inhibition of in vivo angiogenesis. We hypothesize that this association of two different features is probably to avoid an induction of angiogenesis in response to a critical drop of the oxygen supply caused by an ANG II-induced vasoconstriction. The anti-angiogenic action of the AT1 subtype receptor ensures that a regulation of the perfusion pressure by normal ANG II levels is not disturbed by an ischemia-driven formation of new capillaries. We further hypothesize that the proangiogenic receptor pathway through the AT2 subtype receptor diminishes the anti-angiogenic effect by AT1 receptor subtype in order to enable physiological angiogenesis. This hypothesis is supported by findings of an increased expression of AT2 receptor subtypes in the embryonic growth phase (8)
, endometrial proliferation (9)
, or wound healing (10)
. Finally, the present study provides the first evidence for a humoral regulatory pathway of peripheral angiogenesis and establishes a novel rationale for application of drugs interfering with ANG II or its receptor subtypes. Our work should stimulate investigations into the molecular mechanisms of intracellular signaling of the AT1 and AT2 receptor subtypes and the functional implication of the AT2 receptor subtypes in tumor progression or myocardial ischemia whose prognosis depends on the ability to induce neo-vascularization.
Received for publication March 10, 2003. Accepted for publication July 7, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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